Histochemistry and Special Stains

1. Saikat Mitra
Junior Resident
Dept. of Pathology
SGPGIMS

4. Histochemistry

5. Definition
“Histochemistry can be defined as 'the
identification, localization and
quantification, in cells and tissues and
by chemical or physical tests, of specific
substances, reactive groups and
enzyme-catalysed activities’ ”
Pearse AG. The role of histochemistry in increasing objectivity in histopathology. Postgrad Med
J. 1975

6. Basic Principles of
Histochemistry
 Histochemistry combines the methods of histology
with those of chemistry or biochemistry, to reveal
the biochemical composition of tissues and cells
beyond the acid-base distribution shown by
standard staining methods (Hx & E), without
disrupting the normal distribution of the chemicals.

7. The Goal of Histochemistry
 Presentation of Normal Chemical Distribution: The
substance being analyzed must not diffuse away from its
original site.
 Presentation of Normal Chemical Composition: The
procedure must not block or denature the reactive
chemical groups being analyzed, or change normally non
reactive groups into reactive groups.
 Specificity of the Reaction: The method should be
highly specific for the substance or chemical groups being
analyzed, to avoid false-positive results.
 Detectability of the Reaction Product: The reaction
product should be colored or electron scattering, so that it
can be visualized easily with a light or electron
microscope.

8. Application
 Identify, quantify, and localize
 chemical substances
 gene expression
 biological structures, organelles
 specific cell types
 Clarify cell and tissue structure and
morphology.
 Demarcate functional boundaries.

9. Brief History
Francois-Vincent Raspail (1794-
1878)

10.  Rapidity
 Reproducibility
 Cost-effective

11. Terminologies in Histochemistry
 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:
 Functional groups with non-bonded electrons which
augments the colouring property of chromophores.
 Eg: Ionisable OH group turns tri-nitrobenzene into tri-
nitrophenol (Picric acid)

12. Terminologies in Histochemistry
 MORDANTS:
 Non dyeing compound to improve binding of dye
 Mediate a dye –tissue interaction
 Greater stability to stain
 DYE LAKE:
 Dye - mordant complex
 ACCELERATORS:
 To improve staining reactions
 METACHROMASIA:
 Term used when dye stains a tissue component different
color to the dye solution
 Eg: Toluidine blue
 ORTHOCHROMASIA:
 Same color to the dye solution

14. Special Stains for Tissue Types
 Carbohydrate
 Nucleic Acid
 Lipid
 Amyloid
 Stains for Micro-organisms
 Connective Tissue Stains
 Pigments and Minerals

16. Carbohydrates
Simple Carbohydrates
(molecules composed purely of
carbohydrates)
• Monosaccharides
(glucose,mannose,galactose)
• Oligosaccharides
(sucrose,maltose)
• Polysaccharides
(glycogen,starch)
Glycoconjugates
(molecules composed of
carbohydrates and other
molecules such as protein and
lipid)
• Proteoglycans
• Mucins
• Others glycoproteins

17. Proteoglycans
 90-95% of their molecular weight is due to the carbohydrate
component
 The carbohydrate component is known as
glycosaminoglycans(GAG)
 A GAG is composed of repeating disaccharide units , each
made up of 2 different monosaccharides
 Each disaccharide is composed of a carboxylated uronic acid
(glucuronic or iduronic acid) and a hexosamine such as
Nacetylglucosamine or N-acetylgalactosamine
 Types of Glycosaminoglycans
 Chondroitin sulfate
 Dermatan sulfate
 Keratan sulfate
 Heparin sulfate
 Heparin
 Hyaluronic acid

18. Mucins
Neutral
• Stomach, prostate.
PAS +
(Alcian blue colloidal iron and mucicarmine)
-ve
Acid
• Acid (simple, or non-sulfated)
• Epithelial cells containing sialic acid. PAS +Alcian blue + at pH 2.5, colloidal iron +
Resist hyaluronidase digestion. E.g. salivary glands.
• Acid (simple, sulfated -mesenchymal)
• Contain hyaluronic acid, found in tissue stroma. PAS –ve, (Alcian blue at pH 2.5,
colloidal iron, and metachromatic dyes) +. Digest with hyaluronic acid. Found in
sarcomas.
• Acid (complex, epithelial)
• Found in adenocarcinomas of colon, breast,ovary, mucoepidermoid carcinoma . PAS +
Alcian blue positive at pH 1, colloidal iron, mucicarmine, and metachromatic stains are
also positive. Resist digestion with hyaluronidase.
• Acid (complex, connective tissue)
• Found in tissue stroma, cartilage, and bone and include substances such as
chondroitin sulfate or keratan sulfate. PAS negative but do stain selectively with Alcian
blue at pH 0.5.

19. Carbohydrate Staining
 Periodic acid schiff (PAS) technique
 Alcian blue method
 Combined alcian blue- PAS techhnique
 Mucicarmine technique
 Colloidal iron technique
 Metachromatic staining
 Iodine staining for glycogen
 Enzymatic digestion technique
 Diastase digestion
 Sialidase digestion
 Hyaluronidase digestion

20. Periodic Acid Sciff Method
 1st histochemical use was
by McManus for
demonstration of mucin
 Reagents –
 Periodic acid
 Schiff reagent
 0.5-1% solution of periodic
acid (oxidant) used for 5-10
minutes
 RESULT –
 Glycogen and mucins :
 Nuclei : blue
Glutaraldehyde as fixative is avoided for PAS stain
magenta

22. Periodic Acid Schiff (PAS) With Diastase
 PRINCIPLE
 Glycogen is digested with
certain forms of amylase.
Commercially available
diastase or salivary
amylase from saliva can be
used to digest glycogen in
tissue sections
 RESULTS
 Presence of glycogen will
be evidenced by loss of
staining after enzyme
treatment when compared
to the untreated sections

23. PAS Reactive Cells and Tissue
Components
 GLYCOGEN
 STARCH
 MUCIN
 BASEMENT MEMBRANE
 ALPHA-1 ANTI TRYPSIN INCLUSIONS
 RETICULIN
 FUNGI(CAPSULES)
 PANCREATIC ZYMOGEN GRANULES
 THYROID COLLOID
 CORPORA AMYLACEA
 RUSSELL BODIES
 ZYMOGEN GRANULES IN SALIVARY GLAND

24. Uses of PAS stain
 Fungi: stains fungal cell walls
 Hematopathology: ALL, block positivity for PAS
 Kidney:
 routine evaluation of renal biopsies(basement membrane staining)
 renal cell carcinoma (stains glycogen, removed by diastase)
 Liver:
 routine stain for hepatocytes (PAS without diastase)
 inclusions of alpha-1-antitrypsin disease
 Lung: BAL fluid in pulmonary alveolar proteinosis
 Muscle biopsies: routine stain to demonstrate glycogen
 Pancreas: acinar cell carcinoma (PASD+)
 Skin: eosinophilic globoid bodies (Kamino bodies) in Spitz
nevus are PASD+

25. Uses of PAS stain
 Small intestine:
 Whipple’s disease bacteria
 Testis:
 intratubular germ cell neoplasia and seminoma (PAS+, PASD negative),
but not normal seminiferous tubules
 Tumors:
 adenocarcinoma of various sites (mucin is PASD+)
 alveolar soft parts sarcoma (PASD+ crystalline structures)
 apocrine carcinomas
 basement membrane containing tumors (cylindroma, eccrine
spiradenoma)
 clear cell tumors (stains glycogen)
 glycogen rich/balloon cell melanoma
 granular cell tumor (cytoplasmic granules)
 hyaline globules in renal tumors
 Paget’s disease of breast
 Other: stains malakoplakia, hooklet of hydatid cyst

26. Alcian Blue
 Comprised of copper containing pthalocyanine ring linked to 4
isothiouronium groups – strong bases - account for cationic nature
of the dye
 At pH 2.5 it stains – Acidic mucin that include acid-simple or non-
sulfated and acid –simple mesenchymal mucin.
 At pH 1 it stains acid-complex or sulfated mucin
 At pH 0.5 it stains acid-complex connective tissue mucin.
 It does not stain neutral mucin.
 Reagents :
 Alcian blue
 Aluminium sulfate
 Nuclear fast red
 Results
 Sulfomucin,sialomucin
 Proteoglycans
 Hyaluronic acid
 Nucleus - red
Blue

27. Alcian Blue

28. Mucins
Neutral
• Stomach, prostate.
PAS +
(Alcian blue colloidal iron and mucicarmine)
-ve
Acid
• Acid (simple, or non-sulfated)
• Epithelial cells containing sialic acid. PAS +Alcian blue + at pH 2.5, colloidal iron +
Resist hyaluronidase digestion. E.g. salivary glands.
• Acid (simple, sulfated -mesenchymal)
• Contain hyaluronic acid, found in tissue stroma. PAS –ve, (Alcian blue at pH 2.5,
colloidal iron, and metachromatic dyes) +. Digest with hyaluronic acid. Found in
sarcomas.
• Acid (complex, epithelial)
• Found in adenocarcinomas of colon, breast,ovary, mucoepidermoid carcinoma . PAS +
Alcian blue + at pH 1, colloidal iron, mucicarmine, and metachromatic stains are also
positive. Resist digestion with hyaluronidase.
• Acid (complex, connective tissue)
• Found in tissue stroma, cartilage, and bone and include substances such as
chondroitin sulfate or keratan sulfate. PAS negative but do stain selectively with Alcian
blue at pH 0.5.

29. Combined Alcian Blue- PAS
Technique
 Principle
 Demonstrate presence of
mucin
 Differentiate acid mucin
from neutral mucin
 1st stain all acid mucin with
alcian blue (blue)
 Those acid mucin which
are PAS +ve will not be
stained on PAS reaction
 Only neutral mucin will be
stained(magenta)

30. Mucicarmine
 Active dye molecule is aluminium –
carminic acid complex known as CARMINE
 Carminic acid produced from dried bodies
of female Coccus Cacti insects
 Carmine complex has a positive charge
and so attracts polyanions such as
sialomucins and sulfomucins
 Useful for identification of adenocarcinoma
(especially of GIT)
 Capsule of fungus – Cryptococcus
neoformans is also detected

31. Mucicarmine
 Reagents:
 Southgate’s mucicarmine solution
 Alcoholic hematoxylin
 Acidified ferric chloride solution
 Weigert’s iron hematoxylin solution
 Metanil yellow solution
 Results :
 Acidic mucins – deep rose to red
 Nuclei – black
 Other tissue elements – light yellow

32. A. Cryptococcus
B. Mucoepidermoid Carcinoma
C. Normal Goblet cells

33. Hale’s Colloidal Iron Stain
 Positive staining with hale’s colloidal iron
stain is considered diagnostic feature of
chromophobe renal cell carcionoma and
has been used as discriminatory feature
to differentiate it from other renal
tumour.
 Result
 Acid mucopolysaccharides: blue
 Nuclei: red

36. Nucleic Acid
 Two nucleic acids are :
 DNA ( In the nucleus)
 RNA (In the cytoplasm)
 They consist of : Sugar (Deoxyribose /
Ribose), Phosphate and Nitrogenous base
 Demonstration of Nucleic acids depends
upon either
 Reaction of the dyes with the phosphate groups
 Production of aldehydes from the sugar
(deoxyribose)
 No histochemical methods are available to
demonstrate the nitrogenous base

37. DNA is Demonstrated by
 Feulgen technique ( demonstrate sugar)
 Methyl green pyronin technique (demonstrate
phosphate)
 Acridine orange (by fluorescent method)
 Gallocyanin-chrome alum method
 The last staining method do not separate the 2
nucleic acids (stains both DNA and RNA blue)
and suitable extraction technique must be
used
Demonstrates
Both DNA and
RNA

38. Extraction Techniques
 Digestion Method :
 Pure deoxyribonuclease will digest DNA and
pure ribonuclease will digest RNA
 Chemical Method :
 a) By perchloric acid : To remove RNA –
10% perchloric acid at 4 deg C overnight
 b) Trichloroacetic acid
 c) Hydrochloric acid

39. Feulgen Stain
 SOLUTIONS USED ARE :
 1M HCL acid –
○ Used for acid hydrolysis to break the
purine deoxyribose bond and yield an
aldehyde.
○ Done at 60̊ C (HCL should be
preheated to 60 ̊ C )
○ Time (minutes) depends upon the
fixative used, for carnoy’s and formalin
– 8 minutes used
 Schiff reagent –
○ The aldehydes are then demonstrated
by schiff’s reagent
 Bisulfite solution
 RESULT
 DNA : red-purple
 CYTOPLASM : green

40. Methyl Green Pyronin
Method
 Reagents :
 1.Methyl green
○ Impure dye contains methyl violet
– removed by washing with
chloroform
○ Pure methyl green specific for
DNA
○ NH2 of dye reacts with phosphate
of DNA
 2.Pyronin
○ Binds to any negatively charged
tissue constituent
○ Apart from RNA, binds to acid
mucins and cartilage
 Results –
 DNA : green-blue
 RNA : red

41. Points to Remember
 Most suitable technique for identifying DNA is In-situ
hybridization
 Bouin’s fixative is not suitable as it causes over hydrolysis
of the nucleic acid during fixation
 RNA cannot be demonstrated by feulgen stain because
ribose purine bond is unaffected by hydrolysis/ 1 M HCL
 Control method for the standard feulgen technique is
Naphthoic acid hydrazide (NAH) method
○ DNA is acid hydrolysed by 1M HCL.
○ Aldehydes are coupled with naphthoic acid and then again coupled with diazonium
salt, fast blue B.
○ Results are identical to true feulgen reaction
 Blue thionin-feulgen reaction –
○ Used for studying cancer cell nuclear morphology and ploidy. DNA is stained blue
and cytoplasm remains unstained

43. Lipids
 Simple Lipids
 FATS
 OILS
 WAXES
 Compound Lipids
 c/o fatty acids,alcohol and one more group such
as phosphorus or nitrogen
 Derived Lipids
 Derived from simple or compound lipids by
hydrolysis
○ Cholesterol
○ Bile acids

44. Lipids
 Lipids with melting point below staining
temperature can be stained with fat stains
 So only lipids which are liquid at staining temp.
are stained.
 Those in solid or crystalline state remains
unaffected
 Melting point of a lipid is inversely related to its
fatty acid chain length
 Simple lipid is best demonstrated with fresh
frozen sections
 Best fixative
 Formal calcium (2% calcium acetate +10% formalin)

45. Sudan Black B
 1st Sudan dye was Sudan
3
 Most sensitive of all fat
dyes is – Sudan black B
 Sudans must be dissolved
in organic solvents to
penetrate fats
 Some organic solvents
used are –
 1. 70% ethanol
 2. Isopropanol
 3. Propylene glycol
 4. Triethyl phosphate

46. Sudan Black B
 Sudan Black B has 2 fractions –
 1st stains neutral fats blue-black
 2nd stains phospholipids gray
 This gray reaction can be enhanced as a
bronze dichroism if section is viewed in
polarised light
 It fails to stain crystalline cholesterol,
lecithin and free fatty acids
 Bromine pre treatment converts crystalline
cholesterol to oily derivatives and hence
permeable to Sudan dye

47. Oil Red O
 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
 Reagents:
 0.5% Oil red O - Dextrin solution ---- stain for 20 minutes
 Hematoxylin – counterstain ---20 sec
 Aqueous mounting medium - organic solvents found in synthetic
resinous media will dissolve the fat.
 Disadvantages:
 Fat is relatively liquid. so mounting should be done carefully.
 Some neutral fat my be lost during staining
 Technique sensitive

48. Osmium Tetroxide for
Unsaturated Lipids
REAGENTS:
 1% aq. osmium tetroxide for 1 hr
 Mount in glycerin gelly
 RESULTS:
 Unsaturated lipids –
 Saturated lipids /free cholesterol – do not
react
 NB: Handled carefully as toxic vapour
affect cornea and mucous membranes
brown to black

50. Amyloid
 Extracellular , amorphous , eosinophilic
material
 Composed of protein in an antiparallel ß-
pleated sheet configuration
 In H&E stain , can be confused with hyaline
and fibrinoid substances
 Earliest special stain used for amyloid was
Iodine by Virchow

51. Amyloid
Stain used
Congo Red (gold standard). Sirius Red
Metachromatic Techniques
Crystal violet, Methyl violet, Methyl Green
Polarising Microscopy
Apple green birefringence
Acquired fluorescene method: Basic fluorochrome dye Thioflavine T and S
SAP Scintigraphy: Diagnosis as well as monitoring of progression and
regression of amyloid
Immunohistochemistry: For Typing
Laser-Capture Microdissection (LCM) and Tandem Mass Spectrometry (MS)
Proteomic Analysis = For Typing
DNA sequence analysis = Typing eg ATTR(var)

52. Congo Red Stain
 Acidic dye and composed of 2 identical
halves
 Each half has a phenyl ring bound to a
naphthalene moiety by a diazo group
 2 phenyl groups bound by a diphenyl bond -
gives a linear dye molecule
 It stains amyloid by hydrogen bonding and
other tissue components by electrochemical
bonds
 Electrochemical staining of other tissues can
be suppressed by –
 using alkaline-alcoholic solvents
 using competitive inhibition by salt solution
 2 factors are important to the congo red
amyloid reaction
 Linearity of the dye molecule
 Beta-pleated sheet configuration of the amyloid
 If the spatial configuration of either is altered,
the reaction fails

53. Congo Red Stain
Technique
 Fixation – Not critical
 Solution-
○ 0.5 % Congo red in 50% alcohol
○ 0.2% Potassium Hydroxide in 80% alcohol
 Results-
○ Amyloid - red
○ Nuclei – Blue
Alkaine Congo-red Technique
 High concentration of NaCl is used
 Background electrochemical staining is reduced
 Hydrogen bonding of congo-red to amyloid is
enhanced

54. Points to Remember
 Thickness of section is critical – 8-10 micro meter
is ideal
 Too thin section – show faint red color
 Too thick section – show yellow birefringent
 Other structures giving apple-green birefringence :
 1. neurofibrillary tangles of alzheimer’s
 2. intracellular inclusions seen in adrenal cortical cells
 3. cellulose and chitin
 4. dense collagen
 To differentiate AA and AL amyloid :
 Section pretreatment with trypsin or potassium
permanganate done
 AA amyloid lose their affinity for congo-red but AL amyloid
is resistant

55. Methyl /Crystal Violet
Method
 Methyl violet contains
a mixture of tetra- ,
penta- , and hexa-
methyl pararosaniline
 Amyloid stains
metachromatically
(i.e., a different color
from the dye solution,
in this case red
rather than violet)
with crystal violet

57. Stains for
Microorganisms
Gram Staining for Bacteria
 Reagents :
 (1) Crystal violet stain
 (2) Gram’s iodine solution
 (3) Ethyl alcohol – acetone
solution(decolorizer)
 (4) Acetone-xylene solution
 (5) Basic Fuchsin
 (6) Picric acid, 0.1% in acetone
 RESULTS :
 Gram positive bacteria – blue
 Gram negative bacteria – red
 Nuclei– red
 Other tissue elements - yellow

58. Acid Fast Staining for
Bacteria
 Mycobacteria cannot be demonstrated by
gram’s stain
 possess a capsule containing long chain fatty
acid (mycolic acid) that makes them
hydrophobic
 Can be stained by a strong stain like carbol
fuchsin
 Fatty capsule resist the removal of stain by
acid alcohol solution (acid and alcohol
fastness)
 Mycobacteria are PAS positive due to
carbohydrate content of their cell wall

59. Ziehl Neelson (ZN) stain
 Reagents
 (1) Carbol fuchsin solution
 (2) 1% acid alcohol
 (3) 0.1%Methylene blue solution
 Results
 Acid fast bacilli -bright red
 Other tissue -Pale blue
 Caseous material -very pale grayish
blue
 Blue counterstain may be patchy if
extensive caseation is present
 Avoid over counterstaining – scant
organism can easily be obscured
 Decalcification using strong acids
may destroy acid-fastness –
 so formic acid recommended

60. Modified Fite Technique
 Reagents :
 Carbol fuchsin solution
 5% sulphuric acid in 25%
alcohol
 Methylene blue solution
 Results:
 M.leprae – bright red
 Nuclei and other tissue
elements – pale blue
Xylene-peanut oil used for deparaffinization

61. Warthin Starry Method for
Spirochetes
 Reagents :
 Acetate buffer pH-3.6
 1% silver nitrate
 Results :
 SPIROCHETES –
black
 BACKGROUND –
golden -yellow
It can also be used to demostrate
- Helicobacter pylori in gastric mucosa
- Leptospira organism in renal biosy
- Cat scratch disease associated bacilli on
lymph node biopsy

62. Fungal Stains
 Fungal cell walls are rich in
polysaccharides which can be converted
by oxidation to dialdehydes
 Dialdehydes are then detected by silver
solution

63. Gomori Methenamine Silver
nitrate(GMS) technique
Reagents
 4% chromic acid
 1% sodium bisulfite
 5% sodium Thiosulfate
 0.21% Silver nitrate(stock)
 Gold chloride 0.1% aqueous solution
 Light green solution
Results
 Fungi , Pneumocystis, melanin - Black
 Mucin & Glycogen - dark grey
 Background - Pale green
 Hyphae & yeast form - sharply delineated in
black against green background
Cryptococcus by GMS stain

64. Miscellaneous Stains
 Cresyl violet acetate method for
helicobacter pylori
 Macchiavello’s stain for rickettsia and
viral inclusions
 Lendrum’s phloxine – tartrazine stain for
viral inclusions
 Giemsa stain for parasites

66. Connective Tissues

67. Collagen Fibres
 Masson’s trichrome technique
 Van Gieson’s stain
 Mallory’s Phosphotungstic Acid
Hematoxylin
 MSB Technique
 PAS
 Heidenhain’s Azan stain
 Lillie’s allochrome method
 Luxol fast blue G

68. Masson’s Trichrome technique
Principle:
 The term ‘trichrome stain’ is a general
name for a number of techniques for
selectively demonstration of muscle,
collagen fibers, fibrin, and erythrocytes.
 The general rule in trichrome staining is
that the less porous tissues are colored by
the smallest dye molecule; whenever a dye
of large molecular size is able to penetrate,
it will always do so at the expense of the
smaller molecule.

69. Masson’s Trichrome technique
 Reagents
 1. Weigert’s iron hematoxylin
 2. Acid fuchsin
 3. Glacial acetic acid
 4. Phosphomolybdic acid
 5. Methyl blue
 Result
 Nuclei – Blue/ Black
 Cytoplasm, muscle , RBC →
Red
 Collagen → Blue/green

70. Masson’s Trichrome technique
Applications:
 Demonstrate collagen and muscle in
normal tissue
 Differentiate collagen and Muscle in tumors
 Identify an increase in collagenous tissue
 Indicate fibrotic change in cirrhosis of liver
 Indicate fibrotic change in pyelonephritis
 Distinguish tumors that have arisen from
muscle cells and fibroblasts

71. Factors affecting Trichrome
Staining
 Tissue permeability and dye molecular size
 When protein component of a tissue exposed to a fixative – insoluble
protein network formed
 Structure of the protein network directly related to the staining reactions
 Erythrocyte protein – dense network with small pores
 Muscle cells – larger pores
 Collagen – least dense network and quite porous
 Heat :
 Increases rate of staining and penetration
 pH :
 Low pH ( 1.5 – 3)
○ Bouin’s
○ Zenker’s,
○ Formal-mercury
○ Zinc formalin

72. Factors affecting Trichrome
Staining
 Nuclear Stain for Trichrome
 Iron hematoxylin preferred
 More resistant to acidity of dye solutions
 Alum hematoxylins are decolorized
 Can use Celestin blue- alum hematoxylin sequence
 Effect of fixation
 10% NBF will not yield optimal results
 Treatment of formaldehyde fixed tissue with picric acid /mercuric
chloride solution enhances intensity and radiance of trichrome
 Recommended fixatives are:
○ Bouin’s
○ Zenker’s,
○ Formal-mercury
○ Zinc formalin

73. Van Gieson Technique
 Van Gieson’s mixture of picric acid and acid fuchsin
 The simplest method for the differential staining of collagen.
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
staining of collagen will not occur at higher pH levels
REAGENT :
 Weigert’s iron hematoxylin
 Saturated Picric acid solution
 Acid fuchsin
RESULTS :
 Collagen – bright red
 Nuclei – Blue/Black
 Cytoplasm, muscle, RBC , elastin , reticulin -yellow

74. Demonstration of Muscle
Striations
 Haematoxylin and Eosin and trichrome
methods can demonstrate muscle
striations.
 They may also be stained by using
 Heidenhain Iron Haematoxylin
 Mallory’s Phosphotungstic Acid
Haematoxylin.
 Both these methods will give better
definition of muscle striations than the
trichromes.

75. Mallory’s Phosphotungstinic
Acid
Haematoxylin (PTAH) Reagents
 Potassium permanganate - 5min.
 5% Oxalic acid - to remove excess
permanganate.
 PTAH solution - 12-24 hrs at room
temperature
 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

76. Demonstration of elastic tissue
fibres
 Verhoeff ,
 Orcein,
 Weigert’s resorcin-Fuchsin,
 Aldehyde fuchsin.

77. Verhoff’s Elastic Stain
 Verhoeff's Elastic stain is used to demonstrate pathologic conditions
such as atrophy, breaks, thinning, loss etc. in elastic fibers.
Principle
 The tissue is overstained with a soluble lake of hematoxylin-ferric
chloride-iodine. Both ferric chloride and iodine serve as mordants,
but they also have an oxidizing function that assists in converting
hematoxylin to hematein.
 The mechanism of dye binding is probably by formation of hydrogen
bonds, but the exact chemical groups reacting with the hematoxylin
have not been identified.
 This method requires that the sections be overstained and then
differentiated, so it is regressive
Result
 Elastic Fibres – Bluish Black to Black
 Nuclei - Blue to Black
 Collagen - Red
 Other tissue elements – Yellow

78. Demonstration of Reticular
Fibres
 Reticulin fibres are demonstrated either by
using dyes as means of coloring agent or
by metal impregnation methods.
 Techniques :
○ 1.Gordon and sweet’s method
○ 2. Gomori’s method
 Silver impregnation is the best method
because it provides good contrast enabling
even the finest fibers to be resolved

79. Reticulin Stain-Principle
 Reticulin fibers have little natural affinity for
silver solutions.
 On treatment with potassium
permangenate it produce sensitised sites
on fibers where silver deposition can be
initiated.
 The optimal ph for maximum uptake of
silver ions is 9.0.
 A reducing agent formalin causes
deposition of silver in the form of metal.

80. Reticulin Stain
 Dyes used:
○ Silver nitrate 10%
○ NaOH 10%
○ KMnO4 1% aqu.
○ oxalic acid 5% aqu
○ Iron alum 2.5%
○ Formalin 10%
 Control: Cirrhosis of liver
 Result:
○ Reticular fiber – Black
○ Nuclei- Gray
○ Other elements- According to counterstain used

81. Reticulin Stain in : A. Desmoplastic Medulloblastoma B. Myelofibrosis in Acute
Megakaryoblastic Leukemia

83. Pigment and Minerals
 Endogenous Pigment
 Hematogenous
 Non-hematogenous
 Exogenous Pigment
 Asbestos
 Silica
 Lead
 Carbon
 Artefact Pigment
 Formalin
 Malaria
 Mercury
 Schistosomes

84. Hemosiderin
 Breakdown product of haemoglobin
composed of ferric iron and protein
 Seen as yellow-brown granules
 3 methods for demonstration :
 1.Perl’s prussian blue reaction – for ferric ion
 2. Lillie’s method – for ferrous iron
 3. Hukill and putt’s method – for both ferric
and ferrous iron

85. Perl’s Stain
 Principle
 unmasking of ferric iron in hydroxide
form by dilute HCl
 Prussian Blue Reaction–
 Ferric Hydroxide + Potassium
ferrocyanide = Ferric ferrocyanide
(insoluble blue compound)
 Reagents
 2% aq. Potassium ferrocyanide
 2% HCl
 Counterstain with 1% neutral red or
saffranin
 Results
 Ferric iron –Blue
 Nuclei – Red
Best positive control – Postmortem lung tissue that contains a
reasonable amount of iron positive macrophages (heart failure cells)

86. Von Kossa Method for Calcium
 Principle:
 Tissue sections are treated
with silver nitrate solution
 Calcium is reduced by the
strong light and replaced with
silver deposits, visualized as
metallic silver.
 Purpose:
 Abnormal deposits of calcium
may be found in any area of
the body.
 With the H&E stain, calcium
appear deep blue-purple.

87. Bile Pigment: Modified Fouchet’s
technique
 Demonstrates liver bile pigment
 Most common routine method
 Reagents
 Fouchet ‘s reagent :
○ 25% aq trichloracetic acid
○ 10% aq ferric chloride
 Van Gieson stain :
○ acid Fuchsin + saturated aq picric acid
 Result
 Bile pigment : Emerald to blue green
 Muscle :yellow
 Collagen :red
Other Method: Gmelin Technique

88. Melanin
 Normally occur as light brown to black
granules in substantia nigra,hair , skin and eye
 Found Pathologically throughout the body :
 Benign nevus
 Malignant melanoma
 MELANIN DEMONSTRATED BY :
 Reducing methods :
○ a) Masson fontana silver technique
○ b) Schmorl’s ferric-ferricyanide reduction test
 Enzyme methods – DOPA reaction
 Solubility and bleaching characteristics
 Fluorescent method
 Immunohistochemistry

89. Masson-Fontana Stain
 Argentaffin Reaction
 Reduction of ammoniacal silver solution to
form metallic silver without the use of
extraneous reducer.
 Masson’s method (using Fontana’s
silver solution) rely on melanin’s
argentaffin property
 Melanins are blackened by acid silver
nitrate solution
 Result :
 Melanin – black
 Nuclei - red

90. Schmorl’s ferric-ferricyanide
reduction test
 Schmorl reaction – Melanin reduce
ferricyanide to ferrocyanide with
production of prussian blue in the
presence of ferric salts
 Counterstain : 1% neutral red
 Result :
 Melanin – dark blue
 Nuclei – red
 Bleaching of Melanin Pigment
 Strong oxidizing agents are used

91. Lipofuscins
 “Wear snd Tear” pigment
 Polymers of lipid and
phospholipids in complex
with protein
 Normally found in
 Hepatocytes, cardiac
muscles, testis, ovary
 Demonstration techniques :
 PAS method
 Schmorl Reaction
 Suan Black B
 Long Ziehl-Neelsen Method*
*3 hours in carbol fuchsin at 600 water bath/overnight at room-temp.

92. References
 Bancroft’s Theory and Practice of
Histological Techniques, 7th ed
 Riva MA, Manzoni M, Isimbaldi G, Cesana
G, Pagni F. Histochemistry: historical
development and current use in pathology.
Biotech Histochem. 2014 Feb;89(2):81-90
 Wick MR. Histochemistry as a tool in
morphological analysis: a historical review.
Ann Diagn Pathol. 2012 Jan;16(1):71-8