CONNECTIVE
TISSUE STAINS
TOMSON THOMAS
1
SYNOPSIS
• INTRODUCTION
• TERMINOLOGY
• CONNECTIVE TISSUE STAINS
• SUMMARY
• CONCLUSION
• REFERENCES
2
STAINING
IMPORTANCE:
•Tissue lacks contrast – all the fixed tissue section has similar
refractive index
STAIN BINDING MECHANISM:BONDING TYPE STRENGTH(kcal mole-1)
•Ionic bonds 40-110
•Hydrogen bonds 2-7
•Van der Waals forces 1-2
•Covalent bonds 35-122
•Hydrophobic interactions 4-8
3
DYE:
• Coloured organic compounds that can be selectively
bind to tissues.
• Eg: Benzene derivatives
CHROMOPHORES:
• Any group that makes an organic compound coloured
• Eg: Adding single nitro group with benzene will gives
nitro benzene
AUXOCHROMES:
• Binding group -addition of ionizable group - help
binding to tissues
• Eg: Ionizable OH group turns trinirobenzene into trinitrophenol
(Picric acid)
4
MORDANTS:
• Non dyeing compound to improve binding of dye
• Mediate a dye –tissue interaction
• Greater stability to stain
DYE LAKE:
• Dye - mordent complex
ACCELERATORS:
• To improve staining reactions
METACHROMASIA:
• Tem used when dye stains a tissue component different color to the
dye solution
• Eg: Toluidine blue
ORTHOCHROMASIA:
• Same color to the dye solution5
CONNECTIVE TISSUE STAINS
CONNECTERE – TO BIND
CELLULAR PORTION IN A SURROUNDING FRAMEWORK OF
NON CELLULAR SUBSTANCE
INTER CELLULAR SUBSTANCE:
•FORMED OR FIBROUS TYPE
•AMORPHOUS OR GEL TYPE
6
STAINS
FOR COLLAGEN
FOR MUSCLE
FOR ELASTIC TISSUE
FOR RETICULIN FIBRES
FOR CARBOHYDRATES
FOR AMYLOID
FOR LIPID
FOR DECALCIFIED BONE
MISCELLANEOUS:
FOR MELANIN
FOR CALCIUM
FOR NERVE TISSUE
Broadly classifying stains under
following category…..
7
DEMONSTRATION OF
COLLAGEN
o Masson’s trichrome stain {Masson-1929}
o Van Geison’s stain{Van Gieson 1889}
STAINING REACTION:
•STAINS STRONGLY WITH ACID DYES
•AFFINITY OF CATIONIC GROUPS OF THE PROTEINS FOR THE
ANIONIC REACTIVE GROUP OF THE ACID DYES
8
MASSON'S TRICHROME STAIN
• PRINCIPLE:
Smaller dye molecule will penetrate and stain a tissue element ,but when ever the
larger molecule can penetrate the same element ,the smaller molecule is replaced
by it
• THE THREE DYES :
• Aniline Blue (stains collagen and mucus to blue or blue green)
• Beibrich Scarlet (stains cytoplasm, muscle and keratin to red)
• Weigert's Iron Hematoxylin (stains nuclei blue to black)
9
PROCEDUREWeigert's working hematoxylin - 10min
Blue in running tap water for 5 minutes
Biebrich scarlet solution - 5 min
Rinse in distilled water.
Phosphotungstic/phosphomolybdic acid - 10 min
Aniline blue -5 minRinse in distilled water
1% Acetic acid -1 min
Dehydrate, clear, and coverslip10
RESULTS
Cytoplasm, keratin, – Red
muscle fibers,
RBCs, Fibrin
Collagen, mucous, – Blue
Cartilage, Amyloid,
adult or mature bone
Nuclei – Blue- Black
DISADVANTAGES
•Sections overfixed in formalin can stain poorly
•Chance for over differentiation with acetic acid. (blue staining
appears faded)
.
Phosphomolybdic, phosphotungsic acid powders, and acetic acid solutions are skin
and eye irritants, and strong corrosives
11
VAN GIESON’S STAIN
o REAGENTS:
Van gieson solution(Saturated aqueous picric acid +1% acid fuchsin solution) ; 3-5 min
PRINCIPLE:
Picric acid provides acidic pH.
It forms with dyes a complex which has affinity for collagen.
The low pH is very important(1.5 – 3.0), as selective stainingof collagen will not occur at higher pH levels.
12
RESULTS:
Collagen – deep red
Muscle, – yellow
Nuclei – blue to black
DISADVANTAGES:
Washing in water after van gieson solution should be avoided –colour balance being impaired
The tendency for the red colour to fade ; whatever mounting medium is used
Saturation of picric acid is important
unsaturated picric acid will lead to staining of collagen,
cytoplasm, muscle same colour
13
DEMONSTRATION OF MUSCLE
STRIATIONS
- Haematoxylin and eosin and trichrome methods
can demonstrate muscle striations.
- They may also be stained by using:
MALLORY’S PHOSPHOTUNGSTIC ACID
HAEMATOXYLIN
HEIDENHAIN IRON HAEMATOXYLIN
Both these methods will give better definition of muscle
striations.
14
Potassium permanganate - 5min.
5% Oxalic acid - to remove excess permanganate.
PTAH solution - 12-24 hrs at room temperature
REAGENTS:
PRINCIPLE:Referred to as a polychrome stain because one solution gives two
major colors. The hematoxylin lake stains selected tissue components
blue, while the PTA is thought to stain the red-brown components.
MALLORY’S PHOSPHOTUNGSTIC ACID
HAEMATOXYLIN
15
RESULTS:Muscle striations - dark blue
Collagen,osteid,cartilage,elastic fibres - brownish red
DISADVANTAGES:
•Technique sensitive:
Dehydration must be rapid as the components coloured red
brown will lose this color with prolonged water or alcohol wash
16
HEIDENHAINS’S IRON
HEMATOXYLIN
• REAGENTS:
5% Iron alum - 1 hr
Heidenhain’s hematoxylin - 1hr
Heidenhain’s hematoxylin (0.5g Hematoxylin +Absolute alcohol
10ml+ Distilled water 90 ml)
Iron solution is used first ,section then treated with hematoxylin
solution until it is over stained,then differentiated with iron solution
• PRINCIPLE:
Iron alum oxidises hematoxylin into hematein17
RESULTS:
Mitochondria,muscle striations,myelin,chromatin
: gray – black
Cardiac Muscle, Cross section, Heidenhain's iron hematoxylin, 50x
DISADVANTAGE :
Results depend on the degree of differentiation
18
DEMONSTRATION OF ELASTIC TISSUE
FIBRES
. VERHOEFF’S STAIN
ORCEIN STAIN
WEIGERT’S RESORCIN-FUCHSIN
ALDEHYDE FUCHSIN
19
PRINCIPLE:
• Elastin and preelastin are highly crosslinked by
disulphide bridges
• Oxidative treatment- break these bridges and is converted
to anionic sulphonic acid derivatives
• Thus capable of selective reactions with basic dye
compounds
20
VERHOFF’S ELASTIC STAIN
REAGENTS:
• Verhoeff elastic stain ( hematoxylin, ferric chloride, Lugol
• iodine)
• 2% aquous Ferric chloride
• Van Gieson solution (acid fuchsin, saturated picric acid);
.
21
PROCEDURE
Verhoeff's solution - 15-30 min
Differentiate in 2% ferric chloride solution, check
microscopically for black fibers on a gray background
Hypo for 1 min - to remove iodine.
Counterstain in Van Gieson's - 5 min
Dehydrate, clear in xylene, and coverslip
Wash in tap water.
Rinse in water.
Wash in water
22
PRINCIPLE
• The elastic tissue -strongest affinity for the iron-hematoxylin complex
and will retain the dye longer than the other tissue elements.
RESULTS : Elastic Fibres - Bluish Black to Black
Nuclei - Blue to Black
Other tissue elements - According to counter stain
DISADVANTAGES:
•Slides must be individually differentiated as time of
differentiation is dependent on amount of elastic tissue
23
WEIGERT’S RESORCIN-FUCHSIN
STAIN
PRINCIPLE:
• Acetylation,sulphation,phosphorylation -binding of
resorcin-fuchsin to tissues.
• Thus pretreatment – formation of ester group-enable
binding
REAGENTS:
• 1% potassium permaganate followd by oxalic acid – 5min
• 1g basic fuchsin+ 2g resorcin+ 30% ferric chloride
solution ; 1-3 hrs
• Van gieson ; counter stain24
• RESULTS:
Elastic fibre tissue - Brown to purple
Collagen - Pink to redDISADVANTAGES:
Preparation of staining solution is time
consuming
25
ORCEIN STAIN
• Naturally occuring vegetable dye
• Advantage: Simplicity of stain preperation
• REAGENTS:
0.5% periodic acid ; 5min
Orcein : 30 min
• PRINCIPLE:
Van der waals forces between the elastin & orcein.
26
RESULTS:
Elastic fibers – dark brown
DISADVANTAGES:
Less intense color than verhoeff
27
ALDEHYDE FUCHSINGomori 1950
REAGENTS:•1% potassium permaganate - 5min
•1% oxalic acid –to remove excess potassium permaganate
•Aldehyde fuchsin solution (1g basic fuchsin+70% alcohol+1ml conc
HCL+2ml paraldehyde) – 15 min
•Counter stain
RESULTS:
• Elastic fibers - deep blue to dark purple (basic fuchsin)
DISADVANTAGES:
Staining potential diminish after 4 days
28
DEMONSTRATION OF RETICULAR FIBRES:
•FINE AND DELICATE FIBRES
•FOUND CONNECTED TO TYPE I
•WEAK REACTION ATTRIBUTED TO PHYSICAL SIZE
•METAL IMPREGNATION TECHNIQUES ENABLE FINEST FIBRE TO
BE RESOLVED
GORDON AND SWEET’S METHOD,
GOMORI’S METHOD,
29
GENERAL• RETICULAR FIBRES HAVE ONLY LOW AFFINITY FOR SILVER
SALTS
• REQUIRE PRETEATMENT:
HEAVY METAL SALTS- FERRIC AMMONIUM SULPHATE
• UNREACTED SILVER WILL BE REMOVED BY SODIUM
THIOSULPHATE
• TONING: CONVERSION OF METALLIC SIVER TO GOLD
IMPREGNATION- INCREASE CONTRAST
30
GORDON AND SWEET'S METHOD
REAGENTS
1% potssium permaganate solution+ bleach in 1% oxalic acid
2.5% iron alum
Silver solution (10% aq.silver nitrate solution 5ml +
concentrated ammonia + 3% sodium hydroxide 5ml + distilled
water )
0.2% Gold chloride
5% Sodium thiosulfate
Optional counter stain
31
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 gray black, 30 sec.
0.2% Gold chloride, 1 min.
5% hypo, 1 min.
Nuclear-fast red solution, 5 min.32
RESULTS:
Reticular fibres – black
Nuclei - black or unstained
Other elements – according to counter stain
DISADVANTAGES:• Gives much less background and nuclear staining.
• When poor results are obtained ,the stain should be repeated
with particular attention to the diamine silver solution
33
GOMORI STAIN
• REAGENTS:
• 1% potssium permaganate + 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 % aquous formalin - 3 min
• Tone in 0.2% gold chloride 3min
• 5% sodium thiosulphate - 1 min
• Counter stain 34
• RESULTS:• Reticulin : black(silver nitrate)
• Other tissue elements : Counter stain used
DISADVANTAGES:If ammonium hydroxide lost strength,the reticulin may stain
grey black than black
35
RUSSELL MODIFICATION OF
MOVAT PENTACHROME STAIN
• REAGENTS:
Alcian blue - 20 min
Heamtoxylin - 15 min
2% aq.ferric chloride - differentiation
Crocein – sacrlet acid fuschin -5 min
Phototungstic acid – 5 min
Alcoholic sufran - 15 min
• RESULTS:
Elastic fibres - black
Collagen and reticular fibres – yellow
Muscle - red 36
CARBOHYDRATES
Cn(H20)n
37
CLASSIFICATION
38
• Monosaccharide within the tissue specimen are lost during fixation
and tissue processing :
due to the smaller size
water solubility of these molecules
The number of hydroxyl groups present on the monosaccharide
renders most monosaccharides extremely water soluble
39
PROTEOGLYCANS
• HIGHLY GLYCOSYLATED
• 90% IS CARBOHYDRATE COMPONENTS -
GLYCOSAMINOGLYCANS
• Strongly acidic poly anions
• GAG’s – large polysacharide polymers covalently bound
to protein core of proteoglycans
• Negatively charged sulphate and/or carboxyl gps
together with numerous hydroxyl groupings render most
proteoglycans extremely hydrophilic (Accounts for gel
like consistency of the extracellular matrix )
40
41
MUCINS
• Polysaccharide chain covalently linked to a
protein core
• Carbohydrate : 90% molecular wgt
• Polysaccharide chain vary from neutral to weak
acidic
• More varied monosaccharide units than the
GAG’s
• Sialomucins
• Sulphomucins
42
43
STAINS FOR CARBOHYDRATE
o Periodic Acid Schiff Stain
o Alcian Blue
o Mucicarmine Stain – Southgate’s
o Colloidal iron stain
44
PERIODIC ACID SCHIFF (PAS )
McManus 1946
• Widely used technique for the demonstration of carbohydrates or
glycocongugates.
• PRINCIPLE
Based upon the reactivity of free aldehyde groups within
carbohydrates with the schiff reagent to form a bright red
magenta end product
45
46
REAGENTS:
• Periodic acid
• Schiff reagent
(1 gm of basic fuchsin+0.9 g sodium metasulphite+500mg
activated charcoal)
PROCEDURE:Periodic acid for 5 min
Rinse in distilled water.
Schiff's Reagent - 15 min
running tap water for 5-10 min
Counterstain - hematoxylin for 3 minutes.
Dehydrate in alcohol, clear, and coverslip.
47
RESULTS:
Glycogen,neutral/sialomucins - magenta
Nuclei - blue
48
49
PERIODIC ACID SCHIFF (PAS) WITH
DIASTASE FOR THE ENZYME EXTRACTION
OF GLYCOGEN
PRINCIPLE
Glycogen is digested with certain forms of amylase. Commercially
available diastase, which á amylase or salivary amylase from saliva
can be used to digest glycogen in tissue sections.
PAS STAINPAS WITH DIASTASE
ALCIAN BLUE STAIN
Central copper containing pthalocyanin ring linked to four isothiouronium groups via thioether bonds.
Isothiouronium groups- strong bases and hence account for cationic nature
.
• PRINCIPLE
• Exact mechanism unknown;
• Believed that cationic isothiouronium groups bond via
electrostatic linkages with the polyanionic molecules
within tissues
50
• REAGENTS:
• Alcian blue solution(1g Alcian blue + 3% acetic acid)
- 30 min
• Nuclear fast red - Counter stain
• Ph 2.5:
Sialomucins and hyaluronic acid
• Ph 1.0:
• Sulfomucins and sulphate containing proteoglycans
51
RESULTS:
Acid mucins(Sialo and sulphomucins) - blue
Nuclei (using Nuclear fast red) - reddish pink
52
MUCICARMINE STAIN –SOUTHGATE’S 1927
• PAS,AB,Colloidal iron – mucicarmine technique
declined.
• Demonstration of acidic mucin
PRINCIPLE :-
CARMINE – ALUMINIUM CARMINIC ACID COMPLEX.
Aluminium salts form a complex with carminic acid –
conferring an overall positive charge
Attraction to poly anionic substances(Sialomucins and
sulphomucins)
53
REAGENTS:• Carmine (Alum lake) - 1g
• Aluminium hydroxide - 1g
• 50% ethanol - 100ml
PROCEDURE:•Weigert’s hematoxylin - 10 min.
•Mucicarmine solution - 30 min
•Metanil yellow - 30 sec to 1 min.
•Dehydrate quickly in three changes of absolute alcohol, clear and coverslip
RESULTS:
Mucin - deep rose
Nuclei - black
Other tissue elements - yellow 54
COLLOIDAL IRON TECHNIQUE
Hale 1946
PRINCIPLE:
• Based on the attraction of ferric cations in a colloidal
ferric oxide solution for the negatively charged carboxyl
and sulphate groups of acid mucins and proteoglycans.
• As sensitive as alcian blue
• COLLOIDAL IRON WORKING SOLUTION:
29% ferric chloride 4.4 ml+ 250 ml deionized water- boil)
55
REAGENTS:
• 5% Potassium ferrocyanide solution
• 12% acetic acid
• 1% acid fuschin
• Van gieson working solution
RESULTS:
Proteoglycans, hyaluronic acid, acid mucins - Bright blue
Collagen - Red
Muscle - Yellow
56
AMYLOID• Amorphous ,eosinophilic,extracellular material deposited
in various body tissues and organs – amyloidosis
• Virchow(1853)- used iodine reaction – obtained violet
coloration-used the name amyloid
• Gives ‘apple green’ positive birefringence with polarized
microscope
• COMPOSITION:
non fibrilliary glycoprotein known as amyloid P,identical
to serum amyloid P
GAGs-Heparin sulphate, chondroitin sulphate, dermatan
sulphate57
DEMONSTRATION OF AMYLOID
o Crystal Violet
o Congo red
58
CRYSTAL OR METHYL VIOLET STAIN
Hucker & Conn ,1928
PRINCIPLE:
• Affinity of methyl violet to amyloid
• Amyloid from some primary amyloidosis
fail to give positive reaction-Low
sensitivity and lack of specificity
59
• REAGENTS:
CRYSTAL VIOLET SOLUTION: - 5min
2gcrystal violet{Tetra-penta-and hexa pararosaniline}+
80 ml of 1% aqueous ammonium oxalate + 95% alcohol
0.2% acetic acid - differentiate : repeating until good
contrast is obtained.
• RESULTS:
Amyloid,mucin - red-purple
Background - blue
60
CONGO-RED STAIN
Bennholf,1922
•Selective affinity for amyloid
•Congo red – Two phenyl group bound together by
diphenyl bond link – largely hydrophobic
PRINCIPLE:
Hydrogen bonding.
Factors important in dye – amyloid reaction:
Linearity of dye molecule
Beta-pleated sheet configuration.
NB : Prolonged formalin fixation may diminish congo
red staining intensity 61
REAGENTS:
CONGORED SOLUTION: 5 min
• 0.5% congo red in 50% alcohol
• 0.2% potassium hydroxide in 80% alcohol
Alcoholic potassium hydroxide – differentiate ; 5-10 sec
Alum hematoxylin - Stain nuclei
RESULTS:
Amyloid, elastic fibres - Red
Nuclei - Blue 62
POLARISED LIGHT SHOWING “APPLE GREEN”
BIREFRINGENCE OF AMYLOID DEPOSITS63
SIRIUS RED STAIN
• Alternative to congo red.
• Not gained wide acceptance because:
Advantages among congo red are
marginal
No fluorochromatic properties
64
THIOFLAVINE T METHOD
• FLUOROCHROMIC DYE
• ADVANTAGE of not requiring microscopical
differentiation
• Addition of 0.4 M magnessium chloride to 0.1 %
thioflavine T at pH 5.7 – improve selectivity
• Mechanism: Not known
Glomerular Amyloidosis (Thioflavin T Stain)65
LIPIDS
• Defined as anyone of a group of fats or fat like substances
characterized by insolubility in water.
STAINS FOR LIPID:
- OIL RED O
- SUDAN BLACK B
- OSMIUM TETROXIDE
66
OIL RED O
REAGENTS:o 0.5% Oil red O - Dextrin solution ---- stain for 20 minutes
o Hematoxylin – counterstain ---20 sec
Aqueous mounting medium - organic solvents found in synthetic
resinous media will dissolve the fat.
PRINCIPLE :
Frozen or cryostat sections are used.
• Staining with oil-soluble dyes is based on the greater solubility of the dye in the lipid substances
• Inorder to penetrate fats,dye should be dissolved with organic solvents.
• 70% ethanol – adequate solvent
67
RESULTS:
•FAT - RED (OIL RED O).
•OTHER TISSUE ELEMENTS - ACCORDING TO METHOD USED.
OIL RED O STAIN OF FAT EMBOLI IN LUNG.
DISADVANTAGES:
•Fat is relatively liquid. so mounting should be done carefully.
•Some neutral fat my be lost during staining
•Technique sensitive
68
SUDAN BLACK B
PRINCIPLE :
Sudan Black is slightly basic dye and will combine with
acidic groups in compound lipids, thus staining
phospholipids and neutral lipids
REAGENTS:
Saturated Sudan black B +70% ethanol --- 2 hrs
Kernectrot – counterstain --- 2-5 min
RESULTS :
Fat -black
Nuclei -red 69
OSMIUM TETROXIDE FOR
UNSATURATED LIPIDS
REAGENTS:
1% aq. osmium tetroxide for 1 hr
Mount in glycerin gelly
RESULTS:
Unsaturated lipids – brown to black
Saturated lipids /free cholesterol – do not react
NB: Handled carefully as toxic vapour efect-
cornea and mucous membranes70
DEMONSTRATION OF
DECALCIFIED BONE
• H&E staining:
Prolonged decalciifcation
Heat producing methods suchas microwave,
electrolytic method
- will affect the stain
• SCHMORLS’S PICROTHIONIN METHOD
• GOLDNER TRICHROME METHOD
71
SCHMORL’S PICRO-THIONIN
REAGENTS:
• 0.125% aquous thionin – 5 – 20 min
• Saturated aquous picric acid - 30-60 sec
PRINCPLE:
• Depends on the deposition of thionin precipitate
within the lacunae and canaliculi
72
• RESULT
• Lacunae and canaliculi – dark brown-black
• Bone matrix – yellow or brownish
• Cells - red
73
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
74
SOLOCHROME CYANINE
REAGENT:
• Solochrome cyanine R - 1g
• Concentrated sulphuric acid - 2.5 ml
RESULTS:
Mineralized bone - blue
Osteoid - orange
Nuclei - blue
75
MISCELLANEOUS
• STAINS FOR NERVE TISSUE
• STAINS FOR CALCIUM
• STAINS FOR MELANIN
76
STAINS FOR NERVE
TISSUE
• NEURONS:
Cresyl fast violet stain
• NEUROFIBRILS,DENTRITES, AND
AXONS:
Bielschowsky’s stain
• DEGENERATING NERVE FIBRES:
Eager’s method
77
CRESYL FAST VIOLET(NISSL)
STAIN
• Tissue fixed in alcohol stain well
• REAGENTS: Cresyl fastviolet – 0.5 g ; 10-20 min
0.25% Glacial acetic acid ; 10 sec
• RESULTS:Nissl granules - purple
Neurons - Pale purple to blue
NB:0.25 % glacial acetic acid will demonstrate nissl
substance only 78
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:o Silver A
20% silver nitrate
o Reducer A ( make fresh)
Pyrogallol
Formaldehyde
o Silver B
0.2 % gold chloride79
• RESULTS:
Neurofibrils,dendrites and axon - black
80
EAGER’S METHOD FOR
DEGENERATING AXONS
• REAGENTS:
Ammonical silver solution ; 5-15min
1% citric acid+formalin(reducer) ; 2-5min
• RESULTS:
Degenerating fibres - brown to black
Normal fibres - Pale yellow
81
VON KOSSA METHOD FOR
CALCIUMPRINCIPLE:
Tissue sections are treated with silver nitrate solution, the
calcium is reduced by exposing to strong light and replaced
with silver deposits, visualized as metallic silver.
REAGENTS:
1% aquous silver nitrate ; 10 – 30min
1% safran O -Counter stain
RESULTS :
Calcium salts -blackNuclei -red Cytoplasm -pinkCORONARY ARTERY SHOWING CALCIFIED ATHEROMATOUS
PLAQUE82
STAINS FOR MELANIN
o Masson-Fontana silver technique
o Schmorl’s ferric – ferricyanide reduction
test
83
MASSON FONTANA METHOD FOR
MELANIN
PRINCIPLE:
• Melanin is insoluble in organic solvents but soluble
in 1M sodium hydroxide.
• It is slowly bleached by strong oxidising agents.
• The solutions of ammoniacal silver nitrate are
reduced by melanin to black metallic silver
84
ARGENTAFFIN REACTION
Reduction of ammonical silver solutions to form
metallic silver without use of reducer
Melanin is blackened by argentaffin reaction
REAGENTS:
10% aq. nitrate solution ; 30-40 min
0.5% aq. Neutral red ; 5 min
RESULTS:
Melanin : Black
Nuclei : Red
85
86
SCHMORL’S REACTION• PRINCIPLE:
Melanin will reduce ferri cyanide to ferro cyanide with the
production of prussian blue in the presence of ferric salts.
• REAGENTS:
0.4% aqueous potassium ferri cyanide + freshly
prepared aqueous ferric chloride ; 5-10 min
0.5% neutral red ; 5 min
• RESULTS:
Melanin : dark blue
Nuclei : Red87
To summerise…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 •MALLORY PTAH
•HEIDENHAIN’S IRON
HEMATOXYLIN
•MASSON’S TRICHROME STAIN
CARBOHYDRATES •PAS
•ALCIN BLUE
•MUCICARMINE
•COLLOIDAL IRON 88
DEMONSTRATION STAINS USED
AMYLOID •CRYSTAL VIOLET
•CONGO RED
•SIRIUS RED
•THIOFLAVIN -T
LIPIDS •OIL RED O
•SUDAN BLACK B
•OSMIUM TETROXIDE
BONE •SCHMORLS PICROTHIONINE
•GOLDNER TRICHROME
•SOLOCHROME
NERVE •NISSL STAIN
•BIELSCHEWSKY STAIN
•EAGER’S STAIN
MELANIN •MASSON FONTANA SILVER STAIN
•SCHMORL’S FERRICYANIDE STAIN
89
CONCLUSION
Connective tissue stains have been used
extensively for diagnosis of tumours of varying
origins.
Understanding these staining techniques not only
aids us in performing of our staining procedures
effectively but also can facilitate the innovation
of new methods.
90
REFERENCES
• Bancroft JD.Theory and Practice of Histological
techniques; 6th edition Elsevier,China 2008.
• Internet sources
91
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