CONNECTIVE TISSUE STAINS

S.Siva shankari
Department of Oral pathology
 CLASSIFICATION OF CONNECTIVE TISSUE

CONNECTIVE TISSUE

Supporting connective
Connective tissue proper Fluid connective tissue tissue

Blood Lymph
Loose Dense
Cartilage Bone
1)Areolar
1)Hyaline
2)Adipose
2)Elastic
3)Reticular
3)Fibrous
CONSTITUENTS OF CONNECTIVE TISSUE

CELLULAR EXTRA CELLULAR

RESIDENT TRANSIENT FIBRILLAR NON FIBRILLAR

CELL CELL
POPULATION POPULATION
• COLLAGEN
• FIBROBLASTS FIBERS
• LYMPHOCYTES

• MACROPHAGES • PLASMA CELLS

GROUND
• MAST CELLS • RETICULAR SUBSTANCE
• NEUTROPHILS
FIBERS
• ADIPOSE CELLS • EOSINOPHILS

• UNDIFFERENTIAT • BASOPHILS
ED • ELASTIC FIBERS
MESENCHYMAL • MONOCYTES
CELLS
CONNNECTIVE TISSUE STAINS

Collagen fibres
Elastic fibres
Reticulin fibres
Muscle fibres
Carbohydrates
Amyloid
Lipid
Decalcified bone
Nerve tissue
 CONNECTIVE TISSUE STAINS
DEMONSTRATION STAINS USED
Collagen fibres •Masson’s trichrome
•Van gieson

Elastic fibres •Verhoeff’s stain

•Orcein stain
•Weigart’s fuchsin stain
•Aldehyde fuchsin
Reticular fibres •Gordon & sweet’s stain
•Gomori’s stain

Muscle fibres •Mallory PTAH

•Heidenhain’s iron
hematoxylin
•Masson’s trichrome
CONNECTIVE TISSUE STAINS

DEMONSTRATION STAINS USED

Bone •Schmorls picrothionine
•Goldner trichrome
•Solochrome

Nerve •Nissl stain

•Bielschewsky stain
•Eager’s stain
FIBRES OF CONNECTIVE TISSUE
Type Collagen fibres Elastic fibres Reticular fibres

Location In tendons, In skin and walls In spleen, lymph

ligaments, of blood vessels. nodes and bone
bone and marrow.
cartilage

Occurrence Occur in bundles Single Single

Colour Glistening white Yellow White

Nature Unbranched, Branched, Branched and form

thick, long and thin, long and network,
wavy straight. short

Protein Collagen Elastin Reticulin

Elasticity Tough and Elastic Delicate
inelastic
FIBRES OF CONNECTIVE TISSUE
DERMIS OF SKIN

SPLEEN
COLLAGEN SYNTHESIS
 TYPES OF COLLAGEN
Collagen Types Tissue Light Electron Function
distribution microscope microscope

Fibril I,II,III,V Dermis, Thick,highly Densely packed Resistance to tension,

forming ,XI tendon,bone birefringent,no thick fibrils pressure,
collagen fibrocartilage n-argyrophilic structural maintenance
dentin of expansible organs

Fibril- IX,XII Hyaline Fibril lateral

associated cartilage-type association
collagen IX, tendon,
ligaments-type
XII

Network IV,VIII, Basal lamina Thin, PAS Support of delicate

forming X positive & structures
collagen argyrophilic

Anchoring VII Dermis Not visible Clearly visible Binding cells to

collagen subjacent connective
tissue.
DEMONSTRATION OF COLLAGEN

Principle:
• Stains strongly with acid dyes.

• Affinity of cationic groups of the proteins for the anionic

reactive group of acid dyes.

1) Masson’s trichrome stain (Masson-1929)

2) Van Geison’s stain (Van Gieson -1889)
 MASSON’S TRICHROME STAIN

• PRINCIPLE:

Smaller dye molecule will penetrate and stain a tissue element, but whenever larger molecule

can penetrate the same element the smaller molecule is replaced by it.

THE THREE DYES :

• Aniline Blue (stains collagen and mucus -blue or blue green)
• Beibrich Scarlet (stains cytoplasm, muscle and keratin - red)
• Weigert's Iron Hematoxylin (stains nuclei blue to black)
FACTORS AFFECTING TRICHROME STAINS:
i. Tissue permeability and dye molecular size
ii. Heat
iii. pH
iv. Nuclear stain
v. Effects of fixation
PROCEDURE
Weigerts hematoxylin- 10 min
Rinse in running tap water -5 min

Biebrich scarlet solution - 5 min

Rinse in distilled water

Phosphotungstic/phosphomolybdic acid - 10 min

Aniline blue -5 min

1% Acetic acid -1 min

Dehydrate, clear and coverslip

MASSON’S TRICHROME STAIN

APPLICATIONS
In the diagnosis of fibrotic changes,
neuromuscular diseases and tumours of
muscle origin.
DISADVANTAGES
• Sections overfixed in formalin stain
poorly.
• Chances for over differentiation with
acetic acid. (blue staining appears
faded)
• Phosphomolybdic, phosphotungstic
acid powders and acetic acid solutions
– skin, eye irritants and strong
corrosives.
VAN GIESON’S STAIN
REAGENTS:
 Van gieson solution(Saturated aqueous picric acid +1%
acid fuchsin solution) - 3-5 min

PRINCIPLE:
Picric acid provides acidic pH.
 It forms a complex with dyes which has affinity for
collagen.
 The low pH is very important(1.5 – 3.0), as selective
staining of collagen will not occur at higher pH levels.
 VAN GIESON’S STAIN

DISADVANTAGES:

• Washing in water after van gieson

solution should be avoided – colour
balance being impaired.
• Tendency for the red colour to fade.
• Saturation of picric acid

--unsaturated picric acid will lead to

staining of collagen, cytoplasm, muscle
which appear similar.
 ELASTIC FIBRES
 Resilient property of various organs
 Globular molecule, produced by fibroblasts
 2 unusual amino acids :
 Desmosine
 Isodesmosine

 Components
 Microfibrillar & amorphous

 Tissue distribution:
 Skin, lungs, aorta [highest concentration], liver
TYPES OF ELASTIC FIBRES
Type Description Example
Oxytalan Microfibrillar component without Zonule fibers of
amorphous component eye, dermis

Eluanin Microfibrillar component with small Around sweat glands, in

amounts of amorphous components dermis

Elastin Microfibrillar component with abundant Arteries, lung, elastic

amorphous components ligaments, skin and
bladder
DEMONSTRATION OF ELASTIC FIBRES

 Verhoeff’s stain
 Orcein stain

 Weigert’s resorcin-fuchsin

 Aldehyde fuchsin

PRINCIPLE:
 Elastin and preelastin are highly crosslinked by disulphide
bridges.
 Oxidative treatment- breaks these bridges and converted to
anionic sulphonic acid derivatives.
 Selective reaction with basic dye compounds.

APPLICATIONS
To identify atrophy caused by artherosclerotic changes, evidence of
vascular diseases and vessel invasion by tumours.
 VERHOEFF’S ELASTIC STAIN
REAGENTS:
• Verhoeff elastic stain (hematoxylin,ferric
chloride, Lugol iodine)
• 2% aqueous Ferric chloride

• Van Gieson solution (acid fuchsin,

saturated picric acid)

 DISADVANTAGES:

• Slides must be individually differentiated

as time of differentiation is dependent
on amount of elastic tissue
 PROCEDURE

Verhoeff's solution - 15-30 min

Wash in tap water.

Differentiate in 2% ferric chloride solution, check

microscopically for black fibers on a grey background
Rinse in water.

Rinse in 95% alcohol for 1 min - to remove iodine.

Wash in water

Counterstain in Van Gieson's - 5 min

Dehydrate, clear in xylene and coverslip

 WEIGERT’S RESORCIN-FUCHSIN STAIN
PRINCIPLE:
 Acetylation,sulphation,phosphorylation -
binding of resorcin-fuchsin to tissues.
 Pretreatment – formation of ester group-enable
binding
REAGENTS:
 1% potassium permanganate followed

by oxalic acid – 5min

 1g basic fuchsin+ 2g resorcin+ 30% ferric
chloride solution
- 1-3 hrs
 Van gieson counterstain

DISADVANTAGE
Time consuming
 ORCEIN STAIN
Naturally occuring vegetable dye
Advantage: Simple to prepare
REAGENTS:
0.5% periodic acid -5min
Orcein -30 min
PRINCIPLE:
Van der waals forces between the
elastin & orcein
DISADVANTAGES:
Less intensity when compared
to Verhoeff
DEMONSTRATION OF RETICULAR FIBRES
• Fine and delicate fibres.
• Low natural affinity for silver salts-require pretreatment to enhance the
selectivity of impregnation.
• Metal impregnation –provide contrast- resolve fine fibres.
• Treatment with silver-2 fold effect
1) Submicroscopic sensitised sites are created on reticular fibres.
2) Silver is taken up in unreduced form.

GORDON AND SWEET’S METHOD,

GOMORI’S METHOD.
GORDON AND SWEET'S METHOD
Reagents
 1% potassium permanganate solution+ bleach in 1%
oxalic acid
 2.5% iron alum

 Silver solution (10% silver nitrate solution 5ml +

concentrated ammonia + 3% sodium hydroxide 5ml +
distilled water )
 0.2% Gold chloride

 5% Sodium thiosulfate

DISADVANTAGES:
• Gives less background and nuclear staining.

• Poor results -repeated with attention to the diamine

silver solution
 PROCEDURE
Potassium permanganate solution- 5 minutes.
Wash in water.
5% oxalic acid until clear

Iron alum solution-10 min.

Wash in running tap water
Silver solution -2min

10% formaldehyde solution until grey black- 30 sec.

0.2% Gold chloride-1 min.

5% sodium thiosulphate-1 min.

Nuclear-fast red solution-5 min.

 GOMORI’S METHOD.

REAGENTS:

1% potassium permanganate + bleach in 2% potassium

metabisulphate

2% iron alum - 2 min

Silver solution(10% potassium hydroxide 10ml+10% silver nitrate

40ml+conc.ammonia)

Reduce in 4 % aqueous formalin - 3 min

0.2% gold chloride - 3min

5% sodium thiosulphate - 1 min

Counter stain
 GOMORI’S METHOD
APPLICATIONS
Reticular fibres are common in liver, kidney and spleen.
A cirrhotic liver shows disturbed pattern of reticular fibres
explaining routine use of reticular fibre stains in liver biopsy.
In the diagnosis of neoplasm and early cirrhosis of liver.
DISADVANTAGES:
If ammonium hydroxide loses strength, the reticulin stains
appear grey black rather than black.
DEMONSTRATION OF MUSCLE FIBRES
DEMONSTRATION OF MUSCLE FIBRES

 MALLORY’S PHOSPHOTUNGSTIC ACID

HAEMATOXYLIN (PTAH)
 HEIDENHAIN IRON HAEMATOXYLIN

MALLORY’S PTAH
 Reagents –

Potassium permanganate - 5min.

5% Oxalic acid - to remove excess
permanganate
PTAH solution - 12-24 hrs at room
temperature
 MALLORY’S PHOSPHOTUNGSTIC ACID
HAEMATOXYLIN (PTAH)
PRINCIPLE:
Polychrome stain –
one solution gives two major colors.
Hematoxylin-blue
PTA - red brown
APPLICATIONS
For demonstration of cross striations of
skeletal muscles which may be lost in
certain muscle diseases like
rhabdomyosarcoma.
DISADVANTAGES:
 Technique sensitive:

Dehydration must be rapid as the

components coloured red brown will lose
color with prolonged water or alcohol wash
HEIDENHAIN’S IRON HEMATOXLYIN
 REAGENTS:
5% Iron alum - 1 hr
Heidenhain’s hematoxylin - 1hr
Heidenhain’s hematoxylin (0.5g
Hematoxylin +Absolute alcohol
10ml+ Distilled water 90 ml)

 PRINCIPLE:
Iron alum oxidises hematoxylin into
hematein.
DISADVANTAGE :
Results depend on the degree of
differentiation
DEMONSTRATION OF DECALCIFIED BONE
1)SCHMORL’S PICROTHIONIN METHOD
2)GOLDNER TRICHROME METHOD

SCHMORL’S PICROTHIONIN

REAGENTS:
 0.125% aqueous thionin - 5 – 20 min
 Saturated aqueous picric acid - 30-60 sec

PRINCIPLE:
 Depends on the deposition of thionin
precipitate within the lacunae and
canaliculi
GOLDNER TRICHROME METHOD

REAGENTS:
 Weigert’s iron hematoxylin –

1 hour
 Poneau-fuchsin-azophloxin
solution – 5min
 Phosphomolybdic acid- orange G solution – 20min

RESULTS:
 Mineralized bone - green
 Osteoid - orange –red
 Nuclei - blue
APPLICATIONS:
In bone diseases like hyperparathyroidism.
STAINS FOR NERVE TISSUE
 NEURONS:
Cresyl fast violet stain
- For identifying neurons in tissue section and
assessing neuronal damage by demonstration
of Nissl substance.
 NEUROFIBRILS,DENDRITES, AND AXONS:

Bielschowsky’s stain
 DEGENERATING NERVE FIBRES:

Eager’s method
BIELSCHOWSKY’S SILVER STAIN FOR
NEUROFIBRILS, DENDRITES AND AXONS

PRINCIPLE:
The nerve fibers are sensitized with a silver solution. The sections are
treated with ammoniacal silver, and then reduced to a visible metallic
silver.  
REAGENTS:
Silver A -20% silver nitrate
Reducer A
Pyrogallol
Formaldehyde
Silver B
0.2 % gold chloride
APPLICATIONS:
Used in the identification of neurofibrillary tangles and senile
plaques which are hallmarks of Alzheimer’s disease.
EAGER’S METHOD FOR DEGENERATING AXONS
 REAGENTS:
Ammoniacal silver solution - 5-15min
1% citric acid+formalin(reducer) - 2-5min
 RESULTS:

Degenerating fibres - brown to black

Normal fibres - pale yellow
Stain Demonstr Principle Application Advantage Disadvantage
ation
Masson’s Collagen Smaller dye In the diagnosis of To distinguish • Sections overfixed in
trichro- fibrotic changes, between collagen formalin stain poorly.
me molecule will neuromuscular and muscle in • Chances for over
penetrate and stain diseases and smooth muscle differentiation with
tumours of muscle tumours. acetic acid.
a tissue element, origin • Phosphomolybdic,
but whenever phosphotungstic acid
larger molecule can powders and acetic acid
solutions – skin, eye
penetrate the same irritants and strong
element the corrosives.
smaller molecule is
replaced by it.

Verhoeff Elastic  Elastin and To identify Coarse fibres are Slides must be individually
’s fibres preelastin are atrophy caused by well differentiated differentiated as time of
stain highly crosslinked artherosclerotic but fine fibrils are differentiation is dependent
by disulphide changes, evidence frequently lost on amount of elastic tissue
bridges. of vascular during
 Oxidative diseases and differentiation.
treatment- breaks vessel invasion by
these bridges and tumours.
converted to
anionic sulphonic
acid derivatives
 SUMMARY
Stain Demonstr Principle Application Advantage Disadvantage
ation
Gordon Reticular • Low natural In the diagnosis of Metal • Gives less background
and fibres affinity for silver neoplasm and and nuclear staining.
Sweet’s salts-require cirrhosis of liver. impregnation to • Poor results -repeated
method pretreatment to provides with attention to the
enhance the diamine silver solution
selectivity of contrast to
impregnation. resolve these
• Treatment with fine and
silver-2 fold effect
delicate fibres.

Mallory’ Muscle Polychromatic Demonstration No differentiation Technique sensitive:

s PTAH fibres stain- one solution of cross is required and Dehydration must be
gives two major striations of striations are rapid as the
colors. skeletal muscles easily components coloured
which may be demonstrated. red brown will lose
lost in certain color with prolonged
muscle diseases water or alcohol wash
like
Rhabdomyosa-
rcoma.
 CONCLUSION

Connective tissue stains have been used extensively for

diagnosis of tumours of varying origin. Understanding

these staining techniques helps us in performing staining

procedures more effectively.

REFERENCES
 Bancroft’s Theory and practice of histological technique
– 7th edition
 Culling’s Histological technique – 3rd edition

 Crocker and Burnett – The science of laboratory

diagnosis-2nd edition
 Carleton’s Histological Technique – Drury, Fifth edition

  Color atlas of Histological staining techniques-

Smith,Bruton
 Dako’s guide to special stains ,Sonja Wuff,

http://www.lab.anhb.uwa.edu.au