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

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