ANA 403 histochemistry

Histochemical techniques involved in lipids

Group 1

Calvin Abenga CHS/013/12338

Pretreatments

After sectioning, the process of demonstration should be quickly performed to prevent the auto-
oxidation of lipids that can change their physicochemical properties.

Fixation

Frozen sections or carbowax-embedded sections without fixation could obtain the best
demonstration effect of lipids. However, in the application of histochemistry, fixation is necessary for
the preservation of the fine structure of tissues. Alcohol and other lipid-soluble chemicals cannot be
applied in the fixation of lipids as the lipids could be easily dissolved by alcohol. The best fixative that
is commonly used for lipids is Baker’s calcium formalin (FCa) because phospholipids would be better
preserved in the existence of calcium.

Not all types of lipid demonstration require fixation, as some of the lipids can be demonstrated
without performing any fixation step. They are emphasized below:

For lipids like triglycerides, lecithin, sphingomyelin, unsaturated lipids, cholesterol esters, and
plasmalogen phospholipids, after cryostat sectioning, no post-fixation is required.

For lipids like free fatty acids, glycolipids, and phospholipids, staining of all lipids by the Sudan black
method requires post-fixation of 1 hour by calcium-formol in the refrigerator.

For lipids like phosphoglycerides, staining of cholesterol and cholesteryl esters by perchloric acid
naphthoquinone method requires post-fixation by calcium-formol for at least one week in the
refrigerator.

Fixation of materials containing a high amount of neutral lipid droplets should be avoided as it will
merge the lipid droplets.

Histochemical techniques involved in lipids

(A)Demonstration of lipids with physical methods

(I)Neutral lipids demonstration by Sudan black stain

Lysochromes, such as Sudan black and Oil Red O, are soluble in the lipids of tissues (fat-soluble dye),
which can be applied to neutral lipids demonstration.

Materials and reagents

Sudan black solution

Add 0.7g Sudan black into 100ml propylene glycol gradually and stir thoroughly. Heat the solution to
boil and maintain for several minutes, and then filter the solution with Whatman no. 2 filter paper.
Filter the solution again with glass filter after cooling to room temperature.
Kaiser’s glycerol mounting medium

Gelatin-10ml

Distilled water-52.5ml

Glycerol-62.5ml

Phenol-1.25ml

Procedure

The tissue should be fixed by 10% neutral formalin or Bouin the paraffin embedded and sectioned.
Cryostat sections are also feasible.

Dehydrate the sections with pure propylene glycol for 10-15 minutes.

Stain the sections in Sudan black solution for 10 mins.

Differentiate the sections by passing through 85% propylene glycol.

Rinse the sections gently in fresh distilled water (1-2 minutes).

Stain the sections in nuclear fast red for counterstaining cell nuclei.

Rinse the sections thoroughly in fresh distilled water several times.

Mount the section in a Kaiser’s glycerol mounting medium.

Results

Lipids show blue-black colour, whereas cell nuclei are red.

(II)Neutral lipids demonstration by Oil Red O stain

Materials and reagents

Stock solution-Oil Red O isopropyl alcohol saturated solution (99%)

Working solution. Stock solution 6 portion. Distilled water 4 portion.

Filter the solution with Whatman no. 2 filter paper 10 minutes after mixing thoroughly. This working
solution is stable for 1-2 hours.

Hematoxylin- as used in regular histology staining.

Lithium carbonate (0.05%).

Procedure

Rinse the sections in distilled water.

Stain the sections in Oil Red O working solution for 6-15 minutes.

Rinse the section in 60% isopropyl alcohol to clean the background as needed.

Rinse the sections thoroughly in fresh distilled water.

Stain the sections in hematoxylin for counterstaining cell nuclei.

Immerse the sections in 0.05% lithium carbonate.

Rinse the sections thoroughly in fresh distilled water.

Mount the section in water-soluble medium.

Results

Lipids show red, whereas cell nuclei are blue.

(B)Demonstration of lipids with chemical methods

Osmium is a widely used staining agent for lipids demonstration in light and electron microscopy.

Osmium tetroxide (OsO4) is one of the earliest used lysochromes. It dissolves in fats and can be
reduced by organic materials to elemental osmium, an easily observed black substance. It
aggressively oxidizes many materials, leaving behind a deposit of non-volatile osmium in a lower
oxidation state.

Osmium tetroxide is highly poisonous, even at low exposure levels, and must be handled with
appropriate precaution.

Materials and reagents

Osmium tetroxide solution (1%)

Osmium tetroxide 1g

Distilled water 100ml

Procedure

The tissue should be fixed with 10% neutral formalin, and 10-15µm thick frozen sections are
recommended.

Rinse the sections in fresh distilled water.

Stain the sections in 1% osmium tetroxide solution for 24 hours (keep in dark place).

Rinse the sections thoroughly in fresh distilled water.

Immerse the sections into anhydrous alcohol for several hours.

Rinse the section thoroughly in fresh distilled water.

Mount in a Kaiser’s glycerol mounting medium or dehydrate sections with ethanol, clear with xylene
and mount in a resinous medium.

Results

Lipids stain black, whereas the background shows up gray to brown colour.

(C)Demonstration of hydrophobic or hydrophilic lipids

(I)Bromine-Sudan black method

Materials required

Tissue section, 2.5% aqueous bromine, 0.5% sodium bisulfate, Sudan black, 70% ethanol, and
distilled water.

Procedure

Expose the section to 2.5% aqueous bromine for one hour (in a fume cupboard).

Wash the section well with water. Then, dip the section in 0.5% sodium bisulfate to remove excess
bromine.

Put the section in the Sudan black for 10 minutes.

Transfer the section to 70% ethanol for differentiation.

Wash the section well with water and mount it in glycerine jelly for observation.

Observation

Triglycerides and unsaturated fats will be stained in blue-black colour. Some phospholipids will
appear grey.

(II) Marchi method

This method was originally designed to study the finer details of neuroanatomy and pathological
conditions of the nervous system.

Principle: the oxidation-reduction reaction of osmium tetroxide with lipid droplets results in the
formation of a black coloured product.

Materials required:

Sample tissue, 2.5% potassium dichromate, osmium tetroxide 1%, alcohol 70%, alcohol 90%,
absolute alcohol, and distilled water.

Procedure:

Wash the section by using water to remove the formalin used in fixation. This step is not required if
using unfixed tissue.

Put the section in 2.5% potassium dichromate at 21°C for 24 hours.

Wash the section by using distilled water till the deep yellow colour disappears.

Put the section in 1% osmium tetroxide at 21°C (in dark) for 16-36 hours.

Wash the section well with water to remove all acidity.

Put the section in 70% alcohol for 1-2 minutes.

Transfer the section to 90% alcohol for 1-2 minutes.

Transfer the section to absolute alcohol for 1-2 minutes.

Mount the section on a slide for observation.

Black coloured lipid droplets will be observed in the section.

(D) Demonstration of hydrophobic lipids (storage lipids)

Hydrophobic lipids include waxes, lipofuscin, free fatty acids, cholesterols, and triglycerides. The
techniques to demonstrate hydrophobic lipids are given below:

(I)Perchloric acid naphthoquinone (PAN) method

Materials required: sample tissue, naphthoquinone, sulfonic acid, ethanol, 60% perchloric acid, 40%
formaldehyde, 1% ferric chloride, perchloric acid, distilled water, and slide.

Preparation of reagent: mix naphthoquinone and sulfonic acid in a 1:2 ratio and take 40mg from the
mixture, and then add ethanol (20ml), 60 perchloric acid (10ml), 40% formaldehyde (1ml), and
distilled water (9ml) to the mixture. Use this reagent within 24 hours.

Procedure:

Air-dry the section on the slide.

Put the section in 1% ferric chloride for 4 hours.

Wash the section well in distilled water.

Paint the section carefully with the prepared reagent using a camel-hair brush (brush should be
washed after using every time).

Heat the slide at 70°C for 1-2 minutes. Replenish the section with stain to avoid it from drying.

Put a drop of perchloric acid on the coverglass.

Observation: cholesterol and related steroids will be observed in blue colour.

(II)Nile Blue method

Principle: Nile blue is the composition of two dyes that is blue oxazine and red oxazolone (an
oxidation product of oxazine). Oxazone is a lysochrome that reacts with lipids to give a red to pink
colour.

Materials required: sample tissue, Nile blue, sulfuric acid, and distilled water.

Reagent preparation:

Nile blue solution

Add 0.5g of Nile blue to 99ml of distilled water and mix it well, then add 1ml of sulfuric acid.

Procedure:

Wash the section with distilled water.

Put the section in the Nile blue solution for 20 minutes.

Wash the section well with distilled water.

Mount the section in glycerine jelly.

(E)Demonstration of heterophasic lipids

Heterophasic lipids are also called amphipathic lipids. This group includes phospholipids,
glycosphingolipids, and cerebrosides. Histochemical techniques to demonstrate these lipids are as
follows:

(I)Nile Blue method

(II)PAS method

Principle: periodic acid oxidizes the aldehyde group that reacts with Schiff’s reagent which gives a
magenta-coloured product.

Materials required:

Sample tissue, 98% formic acid, hydrogen peroxide, concentrated H2SO4, 10% aqueous chloramine
T, dinitrophenyl hydrazine, 1 M HCL, 0.5% periodic acid, schiff’s reagent, and distilled water.

Reagent preparation

Performic acid

Mix 98% formic acid (45ml), hydrogen peroxide (4.5ml), concentrated H2SO4 (0.5ml) inside a fume
hood.

Procedure:

Deaminate the section in 10% aqueous chloramine T at 37°C for 1 hour.

Wash the section vigorously with water.

Transfer the section to performic acid for 10 minutes.

Wash the section with water.

Treat the section with 0.5% perchloric acid for 10 minutes.

Wash the section by using distilled water.

Stain the section in Schiff’s reagent for 15 minutes.

Rinse the section by using distilled water followed by washing it with the tap water.

Mount the section in glycerine jelly.

Observation: the heterophasic lipid in the section will be stained in magenta colour.

(F)Demonstration of phospholipids

(I)OTAN method

Principle: osmium-α-naphthylamine chelates with the hydrophilic lipid present in the tissue that
gives it orange to red colour.

Materials required: sample tissue, osmium tetroxide, potassium chlorate, α-naphthylamine, and
distilled water.
Reagent preparation

Osmic tetroxide and potassium chlorate (OsO4-KCLO3) solution

Mix 1% osmic tetroxide and 1% potassium chlorate in 1:3 (v/v) ratio.

Α-Naphthylamine solution

Add a saturated aqueous solution of α-naphthylamine in distilled water at 37-40°C. Then, filter the
solution through Whatman no.2 filter paper.

Procedure:

Treat the slide with the OsO4-KCLO3 solution for 18 hours at room temperature.

Wash the slide well with distilled water.

Treat the slide with the α-naphthylamine solution for 15 minutes at 37°C.

Wash the section with distilled water and mount it in glycerine jelly.

Observation: the phospholipid will be stained in orange to red colour.

(II)Chromatin-acid hematin method

Principle: reaction of divalent chromate with the phospholipids form a chelated product. This
product, when it reacts with acid hematin solution, forms a dark blue to black coloured product.

Materials required: sample tissue, 5% potassium dichromate (K2Cr2O7), hematoxylin, sodium iodate
(NaIO3), 0.25% sodium borate, 0.25% potassium ferricyanide, glacial acetic acid, and distilled water.

Reagent preparation:

Hematoxylin solution

Dissolve 50mg of hematoxylin in 50ml of 0.01% NaIO3. Heat the solution until it starts boiling. Cool
the solution and add 1ml of glacial acetic acid.

Borax ferricyanide

Mix 0.25% sodium borate and 0.25% potassium ferricyanide.

Procedure:

By using 5% K2Cr2O7 chromatize the section at 60°C for 4 hours.

Wash the section well by using distilled water.

Stain the section with hematoxylin solution for 30 minutes at 37°C.

Wash the section well with water.

Put the section in the differentiating solution of borax-ferricyanide for 18 hours at 25°C.

Dehydrate the section and mount it on a slide for observation.

Observation: phospholipids will be observed in dark blue to black colour.

Jinsong Zhou (2017). Lipid histochemistry. Histochemistry. Walter de Gruyter GmbH, Berlin/Boston.

Laboratory techniques, protocols, science. Histochemical techniques to demonstrate lipids.