Immunohistochemistry (IHC) an Application Guide

Immunohistochemistry (IHC) an Application Guide Discover more at abcam.com/IHC 1 Contents Tips for designing a successful IHC experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 Sample preparation and Reagents for sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Tissue treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Antigen retrieval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Buffers and epitope retrieval reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 Permeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 Blocking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 Protein on protein blocking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 Biotin Blocking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Peroxidase blocking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Alkaline Phosphatase (AP) blocking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 Autofluorescence blocking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 Blocking cross reactive antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 Reagents for blocking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9 Immunostaining and Reagents for immunostaining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 Immunostaining kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 Biotin-Streptavidin Detection Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 EXPOSE IHC (micro-polymer) Detection Sytems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 Substrates and Chromogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15 Substrate and chromogen products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16 Mounting media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16 IHC controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17 Tissue slides for IHC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17 IHC counterstains and special stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17 Counterstaining products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17 Special stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18 Secondary antibodies for IHC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18 Optimized IHC secondary antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 Using directly labeled primary antibodies for IHC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 Recommended primary antibodies for IHC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 Selected cancer markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 Selected neuroscience markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21 Selected cardiology markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23 Selected immunology markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24 Discover the Rabbit Monoclonal advantage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25 IHC Worksheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26 Note: Products listed are for research use only Discover more at abcam.com/IHC Tips for designing a successful IHC experiment Immunohistochemistry (IHC) is a method for demonstrating the distribution and location of proteins in tissue sections. Though less sensitive quantitatively than immunoassays such as western blotting or ELISA, it enables the observation of processes in the context of intact tissue. This is especially useful for assessing the progression and best treatment options of diseases such as cancer. In general, the information gained from IHC combined with microscopy provides a valuable perspective that can help make sense of data obtained using other methods. Immunohistochemical staining is accomplished with antibodies that recognize the target protein. Since antibodies are highly specific, the antibody will bind only to the protein of interest in the tissue section. The antibody-antigen interaction is then visualized using either chromogenic detection, in which an enzyme conjugated to the antibody cleaves a substrate to produce a colored precipitate at the location of the protein, or fluorescent detection, in which a fluorophore is conjugated to the antibody and can be visualized using fluorescence microscopy. Although IHC is a relatively straightforward experimental method there are a number of variables that have to be identified and optimized for each individual IHC study. Optimization and standardization of these variables would allow consistent and reproducible results. Some variables that should be considered are given in Table 1. Table 1. Immunohistochemistry variables Variable Antigen Epitope Appropriate Controls Factors to consider Species, expression levels, sample types If mapped – dependence on post-translational modification Positive & negative controls - no primary antibody, isotype control, absorption control, tissue type control Sample preparation Fixed or frozen Fixation Method Perfusion or immersion (with our without freezing) Fixative Formaldehyde, alcohols or acetone (including concentration, pH, temperature, incubation time and diluents) Blocking Reagent Normal serum (serum species is critical depending on secondary antibody), bovine serum albumin, gelatine or non-fat milk, commercial blocking buffers Antigen Retrieval Proteolytic-induced Epitope Retrieval (PIER) or Heat-induced Epitope Retrieval (HIER) Detection Method Direct or indirect (with or without amplification) Detection Complex ABC, LSAB, polymer or micro-polymer Primary Antibody Monoclonal or Polyclonal Secondary Antibody Species and label Labelling Method Chromogenic/enzymatic or fluorescence Label Fluorochromes and spectral properties Chromogens: 3,3 Diaminobenzidine DAB,3-amino-9-ethylcarbazole (AEC), benzidine dihydrochloride (BDHC), 3,3’,5,5’-tetramethylbenzidine (TMB), New Fuchsin, 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) Counterstain Fluorescence: DAPI, DRAQ7™, Nuclear green Chromogenic: hematoxylin and eosin or special stain Mounting Reagent Fluorescence: anti-fade mounting medium Chromogenic: organic/aqueous mounting medium Visualization & Analysis Fluorescence microscope or standard microscope. Analysis by eye or software solutions Discover more at abcam.com/IHC 1 Sample preparation and Reagents for sample preparation Sample preparation is an essential and key step in successful histological techniques. Ensuring appropriate and good sample preparation contributes to producing good quality IHC staining. Fixation Fixation prevents the autolysis and necrosis of excised tissues, preserves antigenicity, enhances the refractive index of tissue constituents and increases the resistance of cellular elements to tissue processing. Tissue processing includes dehydration, clearing of dehydrating agents, infiltration of embedding media, embedding and sectioning of tissues. The choice of fixative to be used is dependent on the antigen, it is valuable to try and maintain standardized fixation conditions in order to produce reproducible staining. A robust and optimized fixation protocol is a critical step in an immunohistochemistry protocol as an antigen that has been inappropriately fixed may not be detected in downstream detection. Some guidelines for the type of fixative to use are given in Table 2. Table 2. Fixation guidelines (Fixatives used for a given antigen) Antigen Most proteins, peptides and enzymes of low molecular weight Delicate tissue Small molecules such as amino acids Blood-forming organs (liver, spleen, bone marrow); connective tissue Nucleic acids Large protein antigens (immunoglobulin) Nuclear morphology Ideal for electron microscopy Fixative Cells/Cytological preparations: 4% Formaldehyde Tissue sections: 10% Neutral-Buffered Formalin (NBF) Bouin’s Fixative 4% Formaldehyde Zenker’s Solution Helly Solution Carnoy’s Solution Ice-Cold Acetone or Methanol (100%) Zinc formalin 4% Formaldehyde-1% Glutaraldehyde Figure. 1: ICC Example of how choice of fixative affects immunostaining patterns: Formaldehyde Methanol Primary: ab7291, mouse monoclonal [DM1A] to alpha-Tubulin, 5ug/ml Secondary: ab150105, donkey anti-mouse IgG Alexa Fluor® 488, 2ug/ml 2 Discover more at abcam.com/IHC Embedding Experimental variables and potential downstream protocols influence the most appropriate method that should be used for sample embedding; in addition some epitopes may not survive fixation or embedding. Embedding of tissue is important in preserving morphology and giving the tissue support during microtomy. Some guidelines for tissue embedding are given in Table 3. Table 3: Embedding guidelines Paraffin embedded Frozen tissue Fixation Pre-embedding Pre/post-sectioning Sectioning Microtome Cryostat Storage Multiple years at room temperature (Note: antigen may change over time) 1 year at -80°C (longer at -190°C) Advantages Preserves tissue morphology Preserves enzyme & antigen function Limitations Overfixation can mask the epitope Formation of ice crystals may negatively affect tissue structure Downstream protocols DNA and RNA for PCR amplification DNA, RNA, free nuclei for FISH or cell (extensive crosslinking prevents extraction of cycle analysis long nucleotide strands, free nuclei for ploidy and cell cycle analysis, cell for flow cytometry) Precautions Duration and intensity of tissue heating should be kept to a minimum as melting temperature of paraffin wax (50-60°C) can be deleterious to staining of some antigens Tissues should not be frozen slowly to prevent formation of ice crystals and tissues should be allowed to warm to cutting temperature (-20°C) in cryostat to avoid shattering Tissue treatment Antigen retrieval The process of sample fixation can lead to cross linking which masks epitopes and can restrict antigen antibody binding. Such masked epitopes can be retrieved using Proteolytic-induced Epitope Retrieval (PIER) or Heat-induced Epitope Retrieval (HIER). In the PIER method enzymes such as Proteinase K, Trypsin and Pepsin are used to restore the binding of an antibody to its epitope. The HIER method utilizes heat from a variety of sources (microwave, pressure cooker, steamer, waterbath or autoclave) to unmask epitopes. The preferred technique for optimal retrieval is dependent on tissue, fixation and/or primary antibody and must be optimized by the histologist. Some antigens can be more efficiently retrieved by a combination of heating and enzyme digestion (e.g. some cytokeratins and immunoglobulin light chains). An initial recommendation for optimization is to test two methods from HIER such as citrate buffer (pH 6) and Tris-EDTA (pH 9) and one or two methods from PIER such as Proteinase K and/or Trypsin. Some suggested guidelines are given in Table 4 but conditions must be optimized for each antigen. Discover more at abcam.com/IHC 3 Table 4. Epitope retrieval guidelines HIER PIER Advantages Gentler epitope retrieval and more definable parameters Useful for difficult to retrieve epitopes pH Citrate buffers of pH 6 are widely used but high pH buffers have been demonstrated to be widely applicable for many antibodies. Optimal pH must be determined in the lab Typically 7.4 Recommended No specific antigens antigens Cytokeratins and immunoglobulins Temperature Approximately 95˚C Typically 37˚C Incubation time 10-20 minutes (20 minutes is common) 5-30 minutes (10-15 minutes is common) Buffer composition Depending on pH required as pH is target dependent (as shown in Figure 2). Popular buffer solutions include Sodium citrate, EDTA and Tris-EDTA Neutral buffer solutions of enzymes such as pepsin, proteinase K or trypsin Precautions The use of heating methods such as microwaves can result in unbalanced epitope retrieval due to hot and cold spots. Rigorous boiling can also lead to tissue dissociation from the slide Enzymatic retrieval can sometimes damage the morphology of the section – concentration and treatment need optimization Note: Antigen retrieval may not be required for frozen sections as cross links are formed through formalin fixation. Figure 2. Effects of pH on heat-induced antigen retrieval in human tissues Emoto et al. (2005) Mechanisms of Heat-induced Antigen Retrieval: Does pH or Ionic Strength of the Solution Play a Role for Refolding Antigens? J Histochem Cytochem 53 (11):1311-21. Fig. 3) 4 Discover more at abcam.com/IHC Buffers and epitope retrieval reagents IHC Buffers Product name Background Reducing Buffer 10x Citrate Buffer pH 6.0 100x Citrate Buffer pH 6.0 10x EDTA Buffer pH 8.0 100x EDTA Buffer pH 8.0 10x PBS buffer 25x PBS Buffer pH 7.6 20x PBS Buffer with Tween 20 25x PBS Buffer pH 7.6 20x PBS buffer with Tween 20 25x TBS pH 7.4 20x TBS with Tween 20 25x TBS pH 7.4 20x TBS-T with Tween 20 10x Tris Buffer pH 10.0 100x Tris buffer pH 10.0 Tween 20 Size/description 50mL 125mL 50mL 125mL 50mL 1L 125mL 125mL 1L 1L 125mL 125mL 1L 1L 125mL 50mL 50mL Product code ab64234 ab64214 ab64236 ab64216 ab64239 ab128983 ab64026 ab64028 ab64246 ab64247 ab64203 ab64204 ab64248 ab64250 ab64222 ab64251 ab128987 Epitope recovery/tissue pretreatments Product name Antigen Retrieval Buffer (100X Citrate Buffer pH 6.0) Antigen Retrieval Buffer (100X Citrate Buffer pH 6.0) Antigen Retrieval Buffer (100X EDTA Buffer, pH 8.0) Antigen Retrieval Buffer (100X EDTA Buffer, pH 8.0) Antigen Retrieval Buffer (100X Tris Buffer, pH 10.0) Antigen Retrieval Buffer (100X Tris Buffer, pH 10.0) Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) Heat Mediated High pH Antigen Retrieving Solution 10x Heat Mediated Antigen Retrieval Solution pH 6.0 10X Tris-HCl Buffer for HIER HistoReveal Pepsin Solution Pepsin Solution Proteinase K Trypsin Enzymatic Pretreatment Trypsin Enzymatic Pretreatment Kit Trypsin Enzymatic Antigen Retrieval Solution Discover more at abcam.com/IHC Size/description Product code 125mL ab93678 250mL ab94674 125mL ab93680 250mL ab94677 125mL ab93682 250mL ab94680 125mL ab93684 250mL ab94681 1L ab972 250mL ab973 50mL ab128986 15mL ab103720 7mL ab64201 15mL/125mL ab128991 4mL ab64220 15mL or 125mL ab128214 7mL ab64205 1.6mL concentrated liquid trypsin and 25ml trypsin buffer ab970 5 ed : ur ct at du Fe ro P HistoReveal (ab103720) HistoReveal (ab103720) gives optimal revelation of target antigens, allowing the use of less primary antibody and giving superior staining at the same time. Image: Cytokeratin 20 being stained in colon tissue (Formalin/PFA-fixed paraffin-embedded sections) following antigen retrieval using HistoReveal. Permeabilization Permeabilization is only required when the antibody needs access to the inside of the cells to detect the protein. These include intracellular proteins and transmembrane proteins whose epitopes are in the cytoplasmic region. Solvents or detergents are typically used for permeabilization. Solvents: Can be used after fixation with crosslinking agent e.g. paraformaldeyhyde. Recommended for cytoskeletal, viral and some enzyme antigens. Detergents: Much milder and will not dissolve plasma membrane, suitable for antigens in the cytoplasm or the cytoplasmic face of the plasma membrane and for soluble nuclear antigens. Table 5. Solvent and detergent guidelines Solvents Detergents Solvents Acetone Methanol Triton or NP-40 Tween 20, Saponin, Digitonin and Leucoperm Comments Fixation will also permeabilize. Fixation can be used to permeabilize but is not always suitable. Use 0.1 to 0.2% in PBS for 10 min only. Use 0.2 to 0.5% for 10 to 30 min. Blocking Protein on protein blocking Blocking with sera is essential to block unspecific absorbance to tissue or to Fc receptors. Using a serum matching the species of secondary antibody is recommended. When performing multiple stains using secondary antibodies from different species it may be necessary to using blocking serum from the species of both secondary antibodies. Blocking Sera Sterile sera Product name Bovine Calf Serum (sterile) Cat Serum (sterile) Chicken Serum (sterile) 6 Description/size 25mL 50mL 25mL Product code ab138477 ab139511 ab138577 Discover more at abcam.com/IHC Product name (continued) Dog Serum (sterile) Donkey Serum (sterile) Goat Serum (sterile) Guinea Pig Serum (sterile) Hamster Serum (sterile) Horse Serum (sterile) Mouse Serum (sterile) Rabbit Serum (sterile) Rat Serum (sterile) Sheep Serum (sterile) Description/size 50mL 50mL 50mL 50mL 50mL 50mL 50mL 50mL 50mL 50mL Product code ab7476 ab138579 ab138478 ab138480 ab139500 ab139501 ab138705 ab138706 ab138328 ab138327 Description/size 25mL 10mL 25mL 10mL 25mL 50mL 10mL 10mL 25mL 20mL 10mL 25mL 10mL 50mL Product code ab7479 ab139724 ab7477 ab139737 ab7475 ab7481 ab7482 ab7483 ab7484 ab139738 ab7486 ab7487 ab7488 ab7489 Non-sterile Sera Product name Bovine Calf Serum Cat Serum Chicken Serum Dog Serum Donkey Serum Goat Serum Guinea Pig Serum Hamster Serum Horse Serum Llama Serum Mouse Serum Rabbit Serum Rat Serum Sheep Serum Biotin Blocking Biotin is present in many tissues but is particularly higher in tissues such as kidney, liver or brain tissue. If an Avidin-biotin based detection system is used, blocking endogenous biotin is recommended. In-house protocols for blocking biotin are available but for a robust and reproducible protocol a biotin blocking reagent or a polymer based detection system is recommended (refer to immunostaining kits and reagents page 10). Protocol recommendations: 1. Block endogenous biotin prior to or after incubation with primary antibody but NOT after incubation with a biotinylated secondary. 2. Wetting sample with blocking buffer and flicking this off prior to avidin-biotin blocking creates a nice wet area on and around section for avidin-biotin block Peroxidase blocking When using an HRP conjugated antibody for detection non-specific background staining may occur due to tissue containing endogenous peroxidase. To check for this, tissues can be incubated with DAB substrate prior to primary antibody incubation and if tissues turn brown, endogenous peroxidase is present and a blocking step is required. Hydrogen peroxide (H2O2) is the most commonly used peroxidase blocking agent. Discover more at abcam.com/IHC 7 Protocol recommendations: 1. Block at preferred stage of IHC protocol (i.e. after rehydration to water/before antigen retrieval, after antigen retrieval/before primary incubation, after primary incuation/before secondary incubation or after secondary antibody incubation (for certain antigens like CD4 and CD8 blocking after primary and secondary incubation is recommended as hydrogen peroxide is detrimental to epitopes). 2. Incubate section typically in 0.3% hydrogen peroxide for 10-15 minutes (incubation time may be 5-60 mins depending on concentration of hydrogen peroxide). Alkaline Phosphatase (AP) blocking Endogenous alkaline phophatase (AP) can produce a high background when using AP chromogen substrates. Endogenous AP can typically be found in kidney, intestine, osteoblasts, lymphoid tissue and placenta. Endogenous AP is often more prevalent in frozen tissue and blocking is recommended. Tissue can be tested for endogenous AP through incubation with BCIP/NBT, if a blue colour is observed endogenous AP is present and blocking is necessary. Protocol recommendations: 1. Endogenous AP can be blocked by including levamisole in the chromogen substrate. Chromogens containing levisamole are typically available commercially. 2. For intestinal tissue AP blocking is recommended by treatment with a weak acid prior to application of the primary antibody. Autofluorescence blocking When using a fluorescent label for detection there is a possibility that the unprocessed or fixed tissue maybe autofluorescent. Test samples should be carried out to ensure that the tissue being studied is not inherently fluorescent or that fixation steps do not induce autofluorescence. Potential methods to reduce autofluorescence are outlined below: Table 6. Autofluorescence blocking guidelines Method 1 Method 2 Method 3 Using frozen tissue sections to reduce possibility of induced autofluorescence during fixation Reduce aldehyde presence during fixation by treating tissue with sodium borohydride or glycine/lysine Treating tissue with quenching dyes such as Pontamine sky blue, Sudan black or Trypan blue or FITC block If no solution is found to autofluorescence the use of an enzymatic detection system may be preferable. Blocking cross reactive antigens Mouse on Mouse When staining mouse tissues with mouse primary antibodies, high background may be observed as endogenous mouse IgG will be detected by the secondary targeting the exogenous mouse antibody. To reduce this background in-house, protocols are available from Abcam but to produce robust and reproducible staining a mouse on mouse kit is recommended. 8 Discover more at abcam.com/IHC • • • • Minimal background Strong staining through polymer based detection Biotin-free detection in biotin enriched tissue Simple and reliable protocol for reproducible experiments ed : ur ct at du Fe ro P Mouse on Mouse detection kit (ab127055) Reduction in background staining of endogenous mouse IgG demonstrated in mouse spleen and mouse colon using the Mouse on Mouse detection kit (ab127055): Reduced endogenous mouse IgG background Negative control image, using Mouse on Mouse detection kit (ab127055) on mouse spleen. (Formalin/PFA-fixed paraffin-embedded sections). Negative control image, using EXPOSE detection kit (ab80436) on mouse spleen. (Formalin/PFAfixed paraffin-embedded sections). Stronger staining with Mouse on Mouse detection system Mouse Pan CK (Clone AE1/AE3) antibody staining Pan CK in mouse colon with the use of ab127055. (Formalin/PFA-fixed paraffin-embedded sections). Mouse Pan CK (Clone AE1/AE3) antibody staining Pan CK in mouse colon using ABC Mouse on Mouse system. (Formalin/PFA-fixed paraffin-embedded sections). Reagents for Blocking Product name Endogenous Avidin/Biotin Blocking Kit Endogenous Avidin + Biotin Blocking System FITC Protein Blocking Agent (PBA) Hydrogen Peroxide Blocking Reagent Hydrogen Peroxide Blocking Reagent Protein Block Protein Block Discover more at abcam.com/IHC Size/description 15mL 15mL Avidin, 15mL Biotin 6mL 125mL 60mL 125mL 60mL Product code ab64212 ab3387 ab128980 ab64218 ab94666 ab64226 ab156024 9 Immunostaining and Reagents for immunostaining Immunostaining relies on the detection of specific antibody-antigen interactions with an antibody that has been tagged with a visible label, typically a fluorescent dye, colloidal metal or an enzyme. The steps in a typical chromogenic and fluorescent immunostaining protocol are shown below: Figure 3. Immunostaining flow diagram 10 Discover more at abcam.com/IHC Immunostaining kits There are a number of different labelling techniques that can be used for immunostaining that use in-direct or direct detection of the target antigen. In-direct methods include the use of an avidin-biotin complex (ABC) or a labeled streptavidin-biotin complex (LSAB). Direct methods include the use of a polymer complex or a micro-polymer complex. The ABC method relies on secondary antibodies that are conjugated to biotin to act as links between tissue bound primary antibodies and an avidin-biotin-peroxidase complex. Figure 4. Avidin-Biotin Complex (ABC) method The LSAB method is similar to the ABC method and uses a biotinylated secondary antibody that links primary antibodies to a streptavidin-peroxidase conjugate. The advantage of the LSAB method is that in comparison to avidin, streptavidin has a more neutral isoelectric point and lacks carbohydrate moieties resulting in less nonspecific tissue binding. An advantage of in-direct detection methods is that due to the large enzyme to antibody ratio there is a degree of signal amplification which provides high sensitivity. Figure 5. Labeled Streptavidin-Biotin (LSAB) method Discover more at abcam.com/IHC 11 Although streptavidin-biotin based detection systems are still widely used there are a number of limitations associated with using these methods. The key challenge with these methods is that the presence of endogenous biotin can lead to significant background staining in certain circumstances (e.g. with kidney or brain tissue), this can be worse when staining frozen sections where levels of endogenous biotin tend to be higher than in paraffin-embedded specimens. Direct based detection methods offer a solution to the challenge of endogenous biotin background as well as offering simpler protocols and comparable if not better staining to ABC methods. In polymer based methods a dextran backbone is utilized to which multiple enzyme molecules and secondary antibodies are attached, the dextran backbone and secondary complex then binds to the respective primary antibody. Figure 6. Polymer method More recently, micro-polymer (or compact polymer) based detection methods are available that eliminate the need for a dextran backbone as the enzyme is polymerized directly onto the secondary antibody which forms a smaller detection complex. The main advantages offered by the smaller detection complex are greater sensitivity through better tissue penetration and an improved signal to noise ratio with no endogenous biotin being stained. Figure 7. Micro-polymer method The use of ready-to-use detection kits offers increased reproducibility and consistency. You can find a range of streptavidin-biotin and micro-polymer based (EXPOSE) detection kits available from Abcam. 12 Discover more at abcam.com/IHC Biotin-Streptavidin Detection Systems Abcam’s catalogue includes a number of kits that utilise the LSAB detection system. Choose HRP Plus detection kits for enhanced sensitivity. Mouse and Rabbit specific kits (anti-polyvalent) Product name Mouse and Rabbit specific HRP/DAB (ABC) Detection IHC Kit Mouse and Rabbit specific HRP/DAB Plus (ABC) Detection IHC Kit Mouse and Rabbit specific HRP/DAB Plus (ABC) Detection IHC Kit Mouse and Rabbit specific HRP/AEC (ABC) Detection IHC Kit Mouse and Rabbit specific HRP/AEC Plus (ABC) Detection IHC Kit Mouse and Rabbit specific HRP AEC or DAB (ABC) Detection IHC Kit Mouse and Rabbit specific AP (ABC) Detection IHC Kit Mouse and Rabbit specific AP (ABC) Detection IHC Kit Mouse and Rabbit specific AP/BCIP/NBT (ABC) Detection IHC Kit Mouse and Rabbit specific AP/Fast Red (ABC) Detection IHC Kit Size/description 15mL 60mL 125mL 15mL 125mL 1L 60mL 125mL 15mL 15mL Product code ab64264 ab93697 ab94698 ab93705 ab93677 ab94669 ab93695 ab94725 ab128966 ab128967 Size/description 15mL 15mL 60mL/125mL 15mL 15mL 60mL/125mL Product code ab64261 ab64260 ab128973 ab128968 ab128969 ab128972 Size/description 15mL 15mL 60mL/125mL 15mL 15mL 60mL/125mL Product code ab64259 ab64258 ab128971 ab128964 ab128965 ab128970 Rabbit specific kits: Product name Rabbit specific HRP/DAB (ABC) Detection IHC Kit Rabbit specific HRP/AEC (ABC) Detection IHC Kit Rabbit specific HRP (ABC) Detection IHC Kit Rabbit specific AP/BCIP/NBT (ABC) Detection IHC Kit Rabbit specific AP/Fast-Red (ABC) Detection IHC Kit Rabbit specific AP (ABC) Detection IHC Kit Mouse specific kits: Product name Mouse specific HRP/DAB (ABC) Detection IHC Kit Mouse specific HRP/AEC (ABC) Detection IHC Kit Mouse specific HRP (ABC) Detection IHC Kit Mouse specific AP/BCIP/NBT (ABC) Detection IHC Kit Mouse specific AP/Fast-Red (ABC) Detection IHC Kit Mouse specific AP (ABC) Detection IHC Kit Note: Abcam ABC detection IHC kits use an LSAB detection system Discover more at abcam.com/IHC 13 EXPOSE IHC (micro-polymer) Detection Sytems Benefit from the advantages of using a micro-polymer/compact-polymer detection system. The range available from Abcam offers you greater sensitivity as well as improved signal to noise ratio. Mouse and rabbit specific kits Product name EXPOSE Mouse and Rabbit specific HRP/DAB detection IHC kit EXPOSE Mouse and Rabbit specific HRP/DAB detection IHC kit EXPOSE Mouse and Rabbit specific HRP/DAB detection IHC kit EXPOSE Mouse and Rabbit specific HRP/AEC detection IHC kit EXPOSE Mouse and Rabbit specific HRP/AEC detection IHC kit EXPOSE Mouse and Rabbit specific HRP/AEC detection IHC kit EXPOSE Mouse and Rabbit specific AP (red) detection IHC kit EXPOSE Mouse and Rabbit specific AP (red) detection IHC kit EXPOSE Mouse and Rabbit specific AP (red) detection IHC kit EXPOSE Mouse and Rabbit specific HRP/DAB or AEC detection IHC kit Size/description 15ml 60ml 125ml 15ml 60ml 125ml 15ml 60ml 125ml 1L Product code ab80436 ab94710 ab94709 ab93686 ab94705 ab94706 ab94734 ab94735 ab94736 ab93702 Rabbit specific kits Product name EXPOSE Rabbit specific HRP/DAB detection IHC kit EXPOSE Rabbit specific HRP/DAB detection IHC kit EXPOSE Rabbit specific HRP/DAB detection IHC kit EXPOSE Rabbit specific HRP/AEC detection IHC kit EXPOSE Rabbit specific HRP/AEC detection IHC kit EXPOSE Rabbit specific HRP/AEC detection IHC kit EXPOSE Rabbit specific AP (red) detection IHC kit EXPOSE Rabbit specific AP (red) detection IHC kit EXPOSE Rabbit specific AP (red) detection IHC kit Size/description 15ml 60ml 125ml 15ml 60ml 125ml 15ml 60ml 125ml Product code ab80437 ab94726 ab94727 ab94361 ab94728 ab94729 ab94737 ab94738 ab94739 Size/description 15ml 60ml 125ml Product code ab94740 ab94743 ab94747 Mouse specific kits Product name EXPOSE Mouse specific AP (red) detection IHC kit EXPOSE Mouse specific AP (red) detection IHC kit EXPOSE Mouse specific AP (red) detection IHC kit 14 Discover more at abcam.com/IHC Substrates and Chromogens When using enzymatic detection chromogen/substrates are catalyzed at the site of an enzymatic label to produce a colored precipitate that can be visualised. The Chromogen to be used is dependent on the enzyme being used. The table below offers guidelines to selecting the appropriate label. Table 7. Substrates and Chromogens for IHC Enzyme Horseradish Peroxidase (HRP) Alkaline Phosphatase (AP) Glucose Oxidase Chromogen/ substrate Color Advantages Disadvantages AEC Red Intense color; contrasts well with blue in double staining Aqueous DAB Brown Intense color; permanent Organic Endogenous peroxidase activity in tissue can lead to false positive staining Mounting media DAB + Nickel enhancer Black Intense color; permanent TMB Blue Intense color; permanent Aqueous BCIP/NBT Blue Intense color Organic Naphthol ASMX phosphate + fast blue BB Blue Less intense, good for double staining Naphthol ASMX phosphate + fast red TR Red Less intense, good for double staining Naphthol ASMX phosphate + new fuchsin Red Intense color NBT Blue No endogenous enzyme activity Discover more at abcam.com/IHC Endogenous alkaline phosphatase activity in tissue can lead to false positives. Fast Red and Fast Blue TR Prone to fading Organic Aqueous Aqueous Organic Low staining intensity. High antibody concentrations needed Organic 15 Substrate and chromogen products Product name AEC Single/Plus AEC Substrate System (Ready to Use) Alkaline Phosphatase chromogen (BCIP/TNBT) Alkaline Phosphatase chromogen (BCIP/NBT) Alkaline Phosphatase Enhancer DAB Enhancer DAB Substrate Kit DAB Substrate Kit Fast-Red Substrate System Permanent Fast-Red Substrate System Liquid Fast-Red Substrate Kit Liquid Fast-Red Substrate Kit StayBlue/AP (Alcohol and Xylene substitute compatible) Stay Green/AP (Alcohol and Xylene substitute compatible) StayRed/AP (Alcohol and Xylene compatible) Steady DAB/Plus Streptavidin Alkaline Phosphatase (Ready to Use) Streptavidin Alkaline Phosphatase (Ready to Use) Streptavidin Peroxidase (Ready to Use) Streptavidin Peroxidase (Ready to Use) Size/description 30mL 125mL 100mL 100mL 250mL 10mL 125mL 60mL 60mL 60mL or 125mL 60mL 125mL 30mL 30mL 30mL 200mL 125mL 60mL 125mL 60mL Product code ab103742 ab64252 ab7413 ab7468 ab671 ab675 ab64238 ab94665 ab128979 ab128992 ab128981 ab64254 ab156428 ab103745 ab103741 ab103723 ab64268 ab128984 ab64269 ab128985 Mounting media Mounting of a specimen is essential to preserve the specimen during immunostaining and storage in addition to enhancing image quality during microscopy. Our standard Mounting media can be used to mount setions that have been stained with either DAB or AEC chromogens. Ultra Plus and Vision Mounting Media can be used with either DAB, AEC or FastRed chromogens Mounting Media for IHC: Product name Aqueous Mounting Medium BrightMount BrigthMount/Plus Mounting Medium Vision Mounting Medium 16 size/description 6mL 25mL 25mL 125mL 125mL Product code ab128982 ab103746 ab103748 ab64230 ab94702 Discover more at abcam.com/IHC IHC controls It is essential to run controls in IHC staining experiments to confirm that the observed staining pattern is true, accurate and reliable. Two types of controls are required for the tissue type: Antigen controls: • Positive control: A section from a cell line or tissue known to express the protein you are detecting. A positive result from the positive control, even if the samples are negative, will indicate the procedure is optimized and working. It will verify that any negative results are valid. • Negative control: A section from a cell line or tissue sample known not to express the protein you are detecting. This is to check for non specific binding and false positive results. Reagent control: This ensures that staining is produced from primary antibody staining the antigen and not from detection system or the specimen. This can be determined by using the detection system with diluent alone and no primary antibody. Tissue slides for IHC Abcam offers a range of over 230 tissue slides in both diseased and normal state that can be used as positive staining controls in immunohistochemistry as well as in in-situ hybridization. Tissue types included in the range include spleen, kidney, angioma and Alzheimer tissues. Further information can be found online at www.abcam.com/tissueslides IHC counterstains and special stains After immunostaining of a tissue a second stain is often used to provide contrast that is valuable in making the primary antibody stain stand out. Counterstains are available for chromogenic or fluorescent detection. Popular counterstains are outlined below: Counterstaining products Common counterstains and their targets. Type Chromogenic Chromogenic Chromogenic Fluorescent Fluorescent Fluorescent Fluorescent Fluorescent Fluorescent Fluorescent Dye Hematoxylin Nuclear fast red (Kernechtrot) Methyl green DRAQ7™ Nuclear yellow Nuclear Green DCS1 Hoechst stain 4’, 6-diamidino-2-phenylindole (DAPI) Propidium iodide Fluorophore-tagged phalloidin Discover more at abcam.com/IHC Target Nuclei Nucleic acids Nucleic acids Nucleic acids Nucleic acids Nucleic acids Nucleic acids Nucleic acids Nucleic acids Filamentous actin Color Product code Blue to violet ab1288990 Red ab128992 Green Red Yellow ab138903 Green ab138905 Blue Blue Red ab14083 Fluorophorespecific - 17 Special stains Immunohistochemistry staining can also utilise special staining kits for staining specific cellular or tissue morphology or structures using light microscopy or fluorescence. ed : ur ct at du Fe ro P Hydroxystilbamidine (ab138870) Hydroxystilbamidine (ab138870) also known as Fluoro-Gold™ is a fluorescent dye that can be injected in vivo with flexible post injection survival times. Hydroxystilbamidine (ab138870) can be used as a retrograde enhancer to label neurons. Secondary antibodies for IHC If using an indirect detection protocol then selecting a secondary antibody is necessary, if not provided with the detection system. Secondary antibodies also provide signal amplification compared to direct detection as more than one secondary antibody will bind to the primary. The secondary antibody should be directed against the species the primary antibody was raised in (i.e. if a primary raised in rabbit has been used an anti-rabbit secondary antibody raised in a species other than rabbit must be used). It is also important that the isotype of your secondary antibody matches your primary antibody. Generally, affinity purified antibodies are the most popular as they provide the lowest amount of non-specific binding. However IgG fractions can also potentially contain very high affinity antibodies and may be useful when an antigen is poorly expressed or in low abundance. Pre-adsorbed secondary antibodies are useful for reducing non-specific background as they are less likely to show species cross-reactivity or to react with endogenous antigens of the species they have been preadsorbed against. The secondary antibody should therefore, be pre-adsorbed against the same species the sample originated from. For example, it is advisable to use a secondary antibody pre-adsorbed against human serum when staining human tissues or cell lines. For more information, please go to www.abcam.com/preadsorption. F(ab’)2 fragment secondary antibodies are recommended for staining of tissues rich in Fc receptors (eg. spleen, thymus, blood etc..) to eliminate non specific binding. F(ab’)2 fragment secondary antibodies as they are smaller and therefore more easily penetrate tissues, are particularily useful for multiple IHC staining. Secondary antibodies can be either enzyme labeled (peroxidase, alkaline phophatase), fluorochrome labeled (FITC, R-PE, Alex-Fluor®) or biotinylated. Abcams catalogue contains a range of biotinylated secondary antibodies for use in ABC (avidin biotin complex) detection sytems. 18 Discover more at abcam.com/IHC Optimized IHC secondary antibodies Product name Biotinylated Goat anti Mouse IgG (H+L) (Ready-To-Use) Biotinylated Goat anti Mouse IgG (H+L) (Ready-To-Use) Biotinylated Goat anti Rabbit IgG (H+L) (Ready-To-Use) Biotinylated Goat anti Rabbit IgG (H+L) (Ready-To-Use) Biotinylated Goat anti Mouse & Rabbit IgG (H+L) (Ready-To-Use) Biotinylated Goat anti Mouse & Rabbit IgG (H+L) (Ready-To-Use) Goat anti Mouse IgG secondary antibody (H+L), pre-absorbed Goat polyclonal to Peroxidase anti-Peroxidase complex / PAP antibody Mouse polyclonal to Peroxidase anti-Peroxidase complex / PAP antibody Description/size Product code 125mL ab64255 60mL ab128976 125mL ab64256 60mL ab128978 125mL ab64257 60mL ab128977 1mg ab64244 1mL ab28054 100µl ab21867 Discover other secondary antibodies at www.abcam.com/secondaries Using directly labeled primary antibodies for IHC An alternative to using a secondary antibody for detection in IHC is to use a directly labeled primary antibody. Directly labeled antibodies are suitable for well expressed antigens, for more poorly expressed antigens a secondary detection step is recommended in order to benefit from amplification from the secondary reagent. Using direct detection the primary antibody can be conjugated to an enzyme such as horse radish peroxidase (HRP) or alkaline phosphatase (AP) or alternatively to a fluorochrome. The benefit of direct detection is an additional incubation step with a secondary reagent is not necessary. An additional and significant benefit of direct detection is that when using fluorochromes for direct detection, is increased flexibility in the design of multiple staining experiments with the wide range of fluorochromes that are available. To benefit from direct detection, discover a wide choice of over 25 fluorescent and enzymatic labels for direct conjugation to your primary antibody in the EasyLink range. Discover more at abcam.com/IHC ed : ur ct at du Fe ro P EasyLink 19 Recommended primary antibodies for IHC Selected cancer markers Species reactivity: Chk, Dfsh, Dm, Hu, Mk, Ms, Rat, Zfsh ed : ur ct at du Fe ro P Antibody description: Mouse monoclonal [PC10] to PCNA Proliferation Marker (ab29) Applications: Flow Cyt, ICC/IF, IHC-Fr, IHC-P, IP, WB PCNA is an auxiliary protein of DNA polymerase delta and is involved in the control of eukaryotic DNA replication by increasing the polymerase's processibility during elongation of the leading strand. Induces a robust stimulatory effect on the 3'-5' exonuclease and 3'-phosphodiesterase, but not apurinic-apyrimidinic (AP) endonuclease, APEX2 activities. Has to be loaded onto DNA in order to be able to stimulate APEX2. The image shows ab29 staining of PCNA in mouse embryonic brain tissue section by Immunohistochemistry (Frozen sections). Species reactivity: Cow, Dog, Hu, Ms, Rat, SHm ed : ur ct at du Fe ro P Antibody description: Mouse monoclonal [PAb 240] to p53 (ab26) Applications: Flow Cyt, ICC/IF, IHC-Fr, IHC-P, IP, WB P53 acts as a tumor suppressor in many tumor types; induces growth arrest or apoptosis depending on the physiological circumstances and cell type. Involved in cell cycle regulation as a trans-activator that acts to negatively regulate cell division by controlling a set of genes required for this process. One of the activated genes is an inhibitor of cyclin-dependent kinases. Apoptosis induction seems to be mediated either by stimulation of BAX and FAS antigen expression, or by repression of Bcl-2 expression. Implicated in Notch signaling cross-over. Isoform 2 enhances the transactivation activity of isoform 1 from some but not all TP53inducible promoters. Isoform 4 suppresses transactivation activity and impairs growth suppression mediated by isoform 1. Isoform 7 inhibits isoform 1-mediated apoptosis. The image shows Mouse bone tissue sections stained with ab29 by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Species reactivity: Hu, Ms, Rat, AGMk, Cow, Dog, Zfsh ed : ur ct at du Fe ro P Antibody description: Rabbit polyclonal to E Cadherin (ab53033) Applications: ELISA, Flow Cyt, ICC/IF, IHC-Fr, IHC-P, IP, WB Cadherins are calcium-dependent cell adhesion proteins. They preferentially interact with themselves in a homophilic manner in connecting cells; cadherins may thus contribute to the sorting of heterogeneous cell types. CDH1 is involved in mechanisms regulating cell-cell adhesions, mobility and proliferation of epithelial cells. Has a potent invasive suppressor role. It is a ligand for integrin alpha-E/beta-7. E-Cad/CTF2 promotes non-amyloidogenic degradation of Abeta precursors. Has a strong inhibitory effect on APP C99 and C83 production. The image shows ab53033 staining of E Cadherin in mouse colon tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). 20 Discover more at abcam.com/IHC Product description Species reactivity Applications Rabbit polyclonal to Aurora B Hu, Ms, Rat, Hm ICC/IF, IHC-Fr, IHC-P, IP, WB Product code Rabbit monoclonal [Y69] to c-Myc Hu, Ms, Rat ICC/IF, IHC-P, IP, WB ab32072 Rabbit polyclonal to CD31 Hu, Ms ICC/IF, IHC-FoFr, IHC-Fr, IHC-FrFl, IHC-P, WB ab28364 (phospho Y1092) Hu, Ms ICC/IF, IHC-FoFr, IHC-P, WB ab40815 Mouse monoclonal [IST-9] to Fibronectin Hu, Ms, Rat, Chk, Cow, Dog, Mk, Pig ELISA, ICC/IF, IHC-Fr, IHC-P, RIA, WB ab6328 Rabbit polyclonal to IGF2 Cow, Hu, Ms, Rat ELISA, ICC/IF, IHC-P, Neut, sELISA, WB ab9574 Rabbit polyclonal to N Cadherin Hu, Ms, Rat, Chk, Cow, Zfsh ICC, ICC/IF, IHC-Fr, IHC-P, WB Mouse monoclonal [CH-19] to pan Cadherin Hu, Ms, Rat, Cat, Chk, Cow, Dog, ab2254 Rabbit monoclonal [EP774Y] to EGFR ab12221 Goat, Gpig, Hm, Pig, Rb, SRat, Snk, Xl, Zfsh Flow Cyt, ICC, ICC/IF, IHC-Fr, IHC-P, WB ab6528 Selected neuroscience markers Species reactivity: Hu, Ms, Rat, Cat, Mmst ed : ur ct at du Fe ro P Antibody description: Rabbit polyclonal to GFAP - Astrocyte Marker (ab7260) Applications: ICC, ICC/IF, IHC-FoFr, IHC-Fr, IHC-P, WB GFAP, a class-III intermediate filament, is a cell-specific marker that, during the development of the central nervous system, distinguishes astrocytes from other glial cells. The image shows immunohistochemistical detection of GFAP antibody - Astrocyte Marker (ab7260) on formaldehyde-fixed paraffin-embedded monkey brain sections. Species reactivity: Hu, Ms, Rat ed : ur ct at du Fe ro P Antibody description: Rabbit polyclonal to TBR1 (ab31940) Applications: ICC, ICC/IF, IHC-FoFr, IHC-Fr, IHC-P, WB T box brain 1 (TBR1) is a member of a conserved protein family that share a common DNA-binding domain, the Tbox. T-box genes encode transcription factors involved in the regulation of developmental processes. Disruption of the mouse TBR1 homolog demonstrated a critical role for TBR1 in early cortical development. TBR1 expression is largely restricted to the cerebral cortex, where during embryogenesis it distinguishes domains that give rise to the paleocortex, limbic cortex, and neocortex. The image shows ab31940 staining of rat brain sections by IHC-Fr. The animal was perfused with 4% paraformaldehyde and further post fixed with 4% paraformaldehyde overnight. Discover more at abcam.com/IHC 21 Selected neuroscience markers (Continued) Species reactivity: Hu, Ms, Rat, Mmst ed : ur ct at du Fe ro P Antibody description: Rabbit polyclonal to TBR2 / Eomes - ChIP Grade (ab23345) Applications: ChIP, ChIP/Chip, ICC/IF, IHC-FoFr, IHC-Fr, IHC-FrFl, IHC-P, WB TBR2 functions as a transcriptional activator playing a crucial role during development. Functions in trophoblast differentiation and later in gastrulation, regulating both mesoderm delamination and endoderm specification. Plays a role in brain development being required for the specification and the proliferation of the intermediate progenitor cells and their progeny in the cerebral cortex. Also involved in the differentiation of CD8+ T-cells during immune response regulating the expression of lytic effector genes.The image shows immunohistochemistical detection of GFAP antibody - Astrocyte Marker (ab7260) on formaldehyde-fixed paraffin-embedded monkey brain sections. IHC-FoFr image of TBR2 staining in 8 week old mouse hippocampus using ab23345. Product description Species reactivity Applications Mouse monoclonal [LB 509] to alpha Synuclein Hu, Rat ELISA, Flow Cyt, IHC-FoFr, IHC-Fr, IHC-P, WB ab27766 Rabbit polyclonal to Axin 2 Hu, Ms, Rat Flow Cyt, ICC/IF, IHC-Fr, IHC-P, WB ab32197 Rabbit monoclonal [E247] to beta Catenin Hu, Ms, Rat, AGMk, Hm, Mcq ICC/IF, IHC-Fr, IHC-P, IP, WB ab32572 Rabbit polyclonal to Doublecortin - Neuronal Marker Hu, Ms, Rat, Cat, Chk, Ql, RMk Product code ICC, ICC/IF, IHC-FoFr, IHC-Fr, IHC-FrFl, IHC-P, WB ab18723 Rabbit polyclonal to Dystrophin Dog, Hu, Ms ICC/IF, IHC-Fr, IHC-P, WB Goat polyclonal to Iba1 Hu, Ms, Rat, Gpig, Pig ICC, ICC/IF, IHC-FoFr, IHC-Fr, IHC-FrFl, IHC-P, WB ab15277 ab5076 Rabbit monoclonal [SP6] to Ki67 Proliferation Marker Hu, Ms, Rat ICC/IF, IHC-FoFr, IHC-Fr, IHC-P, WB ab16667 Rabbit polyclonal to LAMP1 Hu, Ms, Rat, Dog, Hm, Xl, Zfsh ICC, ICC/IF, IHC-Fr, IHC-P, IP, WB ab24170 Chicken polyclonal to MAP2 - Neuronal Marker Hu, Ms, Rat, Cow, CynMk ELISA, ICC/IF, IHC (PFA fixed), IHC-FoFr, Rat monoclonal [12] to Myelin Basic Protein - Hu, Ms, Rat, A lept, Cow, Dog Oligodendrocyte Marker ,Gpig, Pig, Rb, Shp Rabbit polyclonal to Parvalbumin Hu, Ms, Rat, Chk ELISA, ICC/IF, IHC-FoFr, IHC-Fr, IHC-P, IP, WB ab11427 Mouse monoclonal [13C4 / I3C4] to PGP9.5 - Hu, Ms, Rat, Dog, Gpig, Pig, ICC, ICC/IF, IHC-FoFr, IHC-Fr, IHC-P, WB Neuronal Marker Rb, Shp, Zfsh IHC-Fr, IHC-P, WB ab5392 ELISA, IHC-FoFr, IHC-Fr, IHC-P, RIA, WB ab7349 ab8189 Mouse monoclonal [SY38] to Synaptophysin Hu, Ms, Rat, Cow, Hm Flow Cyt, ICC/IF, IHC-Fr, IHC-P, WB ab8049 Rabbit monoclonal [EPR3776] to Vimentin Hu, Ms, Rat, RMk Flow Cyt, ICC, ICC/IF, IHC-P, IP, WB ab92547 22 Discover more at abcam.com/IHC Selected cardiology markers Species reactivity: Hu, Ms, Rat, Gpig ed : ur ct at du Fe ro P Antibody description: Rabbit monoclonal [Y266] to Desmin (ab32362) Applications: Flow Cyt, ICC/IF, IHC - Wmt, IHC-Fr, IHC-P, WB Desmin are class-III intermediate filaments found in muscle cells. In adult striated muscle they form a fibrous network connecting myofibrils to each other and to the plasma membrane from the periphery of the Z-line structures. The image shows immunohistochemical analysis (frozen sections) of mouse skeletal muscle tissue following cardiotoxin injury, staining Desmin with ab32362. Species reactivity: Hu, Ms, Rat, Cow, Dog, Pig, Rb ed : ur ct at du Fe ro P Antibody description: Mouse monoclonal [3-48] to heavy chain cardiac Myosin (ab15) Applications: ELISA, Flow Cyt, ICC, ICC/IF, IHC (PFA fixed), IHC-Fr, IHC-P, WB Cardiac myosin heavy chain (MHC) exists as two isoforms in humans, alpha-cardiac MHC and beta-cardiac MHC. These two isoforms are expressed in different amounts in the human heart. During normal physiology, beta-cardiac MHC is the predominant form, with the alpha-isoform contributing around only 7% of the total MHC. Mutations of the MHC genes are associated with several different dilated and hypertrophic cardiomyopathies. The image shows immunohistochemical staining of paraffin embedded rat heart tissue sections by ab15. Species reactivity: Hu, Ms, Rat, Mk ed : ur ct at du Fe ro P Antibody description: Rabbit polyclonal to MEF2C (ab64644) Applications: ICC/IF, IHC-FoFr, IHC-P, WB MEF2C is a transcription activator which binds specifically to the MEF2 element present in the regulatory regions of many musclespecific genes. It controls cardiac morphogenesis and myogenesis, and is also involved in vascular development. It plays an essential role in hippocampal-dependent learning and memory by suppressing the number of excitatory synapses and thus regulating basal and evoked synaptic transmission. Crucial for normal neuronal development, distribution, and electrical activity in the neocortex, it is also necessary for proper development of megakaryocytes and platelets and for bone marrow B lymphopoiesis. Other functions include B-cell survival and proliferation in response to BCR stimulation, efficient IgG1 antibody responses to T-cell-dependent antigens and for normal induction of germinal center B cells. It may also be involved in neurogenesis and in the development of cortical architecture (by similarity). Isoform 3 and isoform 4, which lack the repressor domain, are more active than isoform 1 and isoform 2.. The image shows immunohistochemical staining of MEF2C in formalin-fixed, paraffin-embedded mouse brain sections with ab64644. Discover more at abcam.com/IHC 23 Product description Species reactivity Rabbit polyclonal to alpha smooth muscle Actin Hu, Ms, Rat, Chk, Cow, Dog, Applications Product code Gpig, Pig ELISA, ICC, ICC/IF, IHC-FoFr, IHC-Fr, IHC-P, WB ab5694 Rabbit monoclonal [EP798Y] to Calponin Hu, Ms, Rat, Dog, Pig ICC/IF, IHC-Fr, IHC-P, WB ab46794 Rabbit polyclonal to SM22 alpha Hu, Ms, Rat, Chk, Cow, Pig ICC, ICC/IF, IHC-Fr, IHC-P, WB ab14106 Rabbit polyclonal to CTGF Hu, Rat, Pig ICC/IF, IHC-Fr, IHC-P ab5097 Rabbit polyclonal to KAT13D / CLOCK - ChIP Grade Hu, Ms, Rat, Hm ChIP, GSA, ICC/IF, IHC-Fr, IHC-P, WB ab3517 Rabbit polyclonal to Myeloperoxidase Hu, Ms, Rat, Mk IHC-FoFr, IHC-Fr, IHC-P ab9535 Mouse monoclonal [HM.11] to Nitro tyrosine - ELISA, IHC-Fr, IHC-P, WB Rabbit polyclonal to Nkx2.5 Hu, Ms IHC-Fr, IHC-P, WB ab35842 Rat monoclonal [E13 161-7] to Sca1 / Ly6A/E Ms ICC/IF, IHC-P ab51317 Mouse monoclonal [1B8] to SM22 alpha Hu, Ms, Cow, Pig, Rb Flow Cyt, ICC/IF, IHC-Fr, IHC-P, WB ab28811 Mouse monoclonal [A6.1] to Thrombospondin Hu, Ms, Rat, Cow, Dog, Hrs, Pig, Shp Flow Cyt, ICC/IF, IHC-Fr, IHC-P, IP, WB ab7048 ab1823 Selected immunology markers Species reactivity: Hu ed : ur ct at du Fe ro P Antibody description: Rabbit monoclonal [EP1347Y] to CD11c (ab52632) Applications: ICC, IHC-Fr, IHC-P, IP, WB Integrin alpha-X/beta-2 is a receptor for fibrinogen. It recognizes the sequence G-P-R in fibrinogen. It mediates cellcell interaction during inflammatory responses and is especially important in monocyte adhesion and chemotaxis. The image shows immunohistochemical staining of human tonsil tissue using ab52632 (formalin/PFA-fixed paraffinembedded sections). Species reactivity: Hu, CynMk, RMk ed : ur ct at du Fe ro P Antibody description: Mouse monoclonal [236A/E7] to FOXP3 (ab20034) Applications: Flow Cyt, ICC/IF, IHC-Fr, IHC-P, WB FOXP3 is a transcription factor that plays a critical role in the control of immune response. Defects in FOXP3 are the cause of immunodeficiency polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) [MIM:304790]; also known as X-linked autoimmunity-immunodeficiency syndrome. IPEX is characterized by neonatal onset insulin-dependent diabetes mellitus, infections, secretory diarrhea, trombocytopenia, anemia and eczema. It is usually lethal in infancy. The image shows immunohistochemical staining of human tonsil tissue using ab20034. 24 Discover more at abcam.com/IHC Selected immunology markers (Continued) Species reactivity: Hu, Rat ed : ur ct at du Fe ro P Antibody description: Mouse monoclonal [AA1] to Mast Cell Tryptase (ab2378) Applications: ELISA, ICC, ICC/IF, IHC-Fr, IHC-P, IHC-R, WB Mast cells contain a number of preformed chemical mediators such as histamine, chymase, carboxypeptidase and proteolytic tryptase. Human Mast Cell Tryptase is considered to be an important marker of mast cell activation as well as an important mediator of inflammation. The image shows immunohistochemical staining of human tonsil tissue using ab2378. Product description Species reactivity Applications Rabbit monoclonal [SP7] to CD3 Hu, Mk, Ms, Rat IHC-FoFr, IHC-Fr, IHC-P, WB Product code Mouse monoclonal [QBEND-10] to CD34 Hu, CynMk, RMk Flow Cyt, IHC-Fr, IHC-P, IP, WB ab8536 Mouse monoclonal [F10-44-2] to CD44 Hu Flow Cyt, ICC/IF, IHC-Fr, IHC-P, IP ab6124 Mouse monoclonal [KP1] to CD68 Hu, Ms ICC/IF, IHC-FoFr, IHC-Fr, IHC-P Rabbit polyclonal to CD274 Hu, Ms, Rat IHC-P, WB ab58810 Mouse monoclonal [EMR8-5] to HLA Class 1 ABC Hu Flow Cyt, ICC/IF, IHC-P, WB ab70328 Mouse monoclonal [NAT] to PD1 Hu Flow Cyt, ICC/IF, IHC-Fr, IHC-P, IP, WB ab52587 Mouse monoclonal [TIA-1] to TIA1 Hu, Rat, Mk IHC-P ab16669 ab955 ab2712 Discover the Rabbit Monoclonal advantage Rabbit monoclonal antibodies (RabMAbs®) combine the superior antigen recognition of a rabbit antibody with the specificity and consistency of a monoclonal. The rabbit immune system generates antibody diversity and optimizes the affinity by mechanisms that are more efficient than those of mice and other rodents. This increases the possibility of obtaining a functional antibody that will work in a variety of applications. The rabbit monoclonal advantages: 1. Low background 2. Ideal for post-translational modification detection 3. Excellent for IHC usage 4. High affinity 5. High specificity 6. Diverse/Novel epitope recognition 7. Fully validated in multiple applications 8. Ideal for use on mouse samples ed : ur ct at du Fe ro P Her RabMAb - 3ng / mL Rabbit polyclonal antibody (Vendor A) - 20ng / mL Discover more at abcam.com/IHC Mouse monoclonal (Vendor B) - 30ng / mL Comparison of a Her2 RabMAb with leading commercially available Her2 rabbit polyclonal (vendor A) and mouse monoclonal (vendor B) antibodies on FFPE human breast carcinoma tissue. 25 IHC Worksheet Sample No. 26 (Photocopy this worksheet to help planning your experiments) Tissue / cell (type, species, disease state, format) Fixation (buffer, concentration, temperature, duration) Discover more at abcam.com/IHC Type of antigen retrieval (if required) Discover more at abcam.com/IHC Antigen retrieval (buffer, pH, composition, duration, temperature) Blocking step(s) (composition, duration, temperature) 27 IHC Worksheet Sample No. 28 (Photocopy this worksheet to help planning your experiments) Primary antibody (diluent, concentration, duration, temperature) Detection system (type, concentration, duration, temperature, label, chromogen) Discover more at abcam.com/IHC Mounting media Discover more at abcam.com/IHC Additional notes 29 Discover more at abcam.com/IHC 013_13_GM Follow us on: