VARIOUS STAINS AND THEIR UTILITY

1. Presented by –Dr Sonal Verma

2. Stains for carbohydrates
Stains for connective tissue
Stains for pigments & minerals
Stains for lipid
special stains

3. Classification-
Monosaccharides-
glucose,mannose,galectose
oligosaccharides-
sucrose, maltose
Polysaccharides
glycogen , starch
Connective tissue glycoconjugates-
Proteoglycans ,hyaluronic acid
Mucins-
neutral mucins , sialomucins ,sulfomusins
Other glycoproteins-
membrane protein, blood group antigen
Glycolipid-
cerebrosides , gangliosides

4. TYPE LOCATION FUNCTION PATHOLOGICAL
CONDITIONS
Glycogen
Liver, skeletal
muscle ,cardiac ms,
hair follicle, cervical
epithelium
storage Ewing’s sarcoma/ PNET,
Rhabdomyosarcoma,
Seminoma
Proteoglycans
and
hyaluronic
acid
Cartilage, heart
valve, blood vessel,
tendon ligaments,
extracellular
matrices
Supportive,
Lubrication
Myxoid chondrosarcomas,
Myxoid liposarcomas,
Myxoid fibrous histiocytomas,
Mucopolysaccharidoses
Mucins Epithelia of
GIT,respiratory
tract, reproductive
tract
Lubrication
Protection
Adenocarcinoma of GIT
Aberrant expression of specific
mucin types in neoplastic
process
Glycoprotein Cell mb, blood group
antigen,
Secerted product like
hormones and
immunoglobulins
Cell
Adhesion
,immune
recognition
Aberrant expression of blood
group antigen in various
malignancy

5. STAINS FOR CARBOHYDRATE
o Periodic Acid Schiff Stain
o Alcian Blue
o Mucicarmine Stain – Southgate’s
o Other- Colloidal iron
High iron diamine

6. PAS staining
Principle:
The PAS stain is a histochemical reaction in which
the periodic acid oxidizes the hydroxyl group of
carbohydrate and result in formation of dialdehyde
which react to schiff reagent and form the magenta
color.
Glucose + Periodic acid - aldehyde (Carbohydrate)
Aldehyde + Schiff’s reagent - magenta
colored compound

7. - Periodic Acid Solution
Periodic acid (1 gm) + Distilled water (100 ml)
- Schiff Reagent
Dissolve 1gm of basic fuchsin and 1.9 gm sodium metabisulphite in
100 ml HCL mix, add 500gm of activated charcoal .
SPECIMEN
Standard paraffin section fixed in 10% neutral buffered formalin.
PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Oxidise with periodic acid for 5 minutes.
3. Rinse in distilled water.
4. Treat with Schiff's reagent for 5 minutes.
5. Wash in running water for 10 minutes; this intensifies the colour
reaction.
6. Stain the nuclei with Mayer's haematoxylin for 1 minute .
7. Wash in water.
8. Dehydrate, clear and mount.

8. Results:
PAS positive
substances:
magenta
Nuclei: blue

9. Glycogen in Ewing Sarcoma Candida Hyphae in chronic
Candidosis

10. PAS positive entities
 Polysaccharides (glycogen)
 Starch
 Mucin
 Basement membrane
 Alpha antitrypsin
 Reticulin
 Fungi
 Pancreatic zymogen granules
 Thyroid colloid
 Corpora amylacea
 Russell body

11. o Diastase and alpha-Amylase are enzymes that digest
glycogen polymer to maltose and glucose that are easily
washed from the sections, thus giving a negative
reaction with PAS.
o So staining loss following diastase digestion is
indicative of glycogen
o While if after digestion PAS stain persists is indicative
of presence of mucins.
PERIODIC ACID SCHIFF (PAS) WITH DIASTASE (PAD)

12. RESULTS
Presence of glycogen will be evidenced by loss of
staining after enzyme treatment when compared to
the untreated sections.
PAS Stain
PAS with Diastase

13. Mucin
Neutral
Found in epithelia of
gastric mucosa,
brunner glands of
duodenum,
prostatic epithelium
Acidic
Sialomucin
1)Enzyme labile: Digested with
hyaluronic acid
bronchial submucous glands,
submandibular gland, goblet cells of
small intestine
2) Enzyme stable: resist
hyaluronidase digestion
Mucosa of large intestine,stomach and
bronchus
Sulfomucin
stroma, cartilage,
malignancy

14.  PAS
 PAD
 Alcian Blue
 Mucicarmine
 High Iron Diamine
 Colloidal Iron

15. Mucin
Neutral
PAS +ve
PAD+ve
AB-Ve,
muc,coll,HID-ve
Acidic
Sialomucin
1)Enzyme labile:
PAS +ve, PAD+ve,
AB+ve, muc+ve,
coll+ve, HID-ve.
2) Enzyme stable:
PAS-ve,PAD-ve
AB +ve, Muc+ve
coll+ve,HID-ve
Sulfomucin
PAS V/-ve,
PA D v/-ve
AB +ve
Muc,coll,HID+ve

16. TYPE OF
MUCIN
LOCATION/FUNCTION IMPORTANCE
MUC 1 Cell surface associated Overexpression in ovarian,lung
,breast,colon and pancreatic cancer
MUC 2 Secreted mucin Prominent in GIT,
secrted from goblet cell
MUC 3 Cell surface associated
MUC 4 Cell surface associated Associted with pancreatic
adenocarcinoma, oesophagial ca,
breast ca,thyroid ca
MUC 5 Secreted mucin Hypersecreted in pulmonary tract and
associated with COPD
MUC 6,7 Secreted mucin

17. PRINCIPLE -
 Alcian blue is a copper containing basic dye
 It contains positively charged groups, capable of salt linkage with certain
polyanions.
 Polyanions of acid mucins -sulphate and carboxyl radicals .
 Thus only the acid mucins are stained.
 pH variation: more information can be gained concerning the types of
mucin present
 Highly acidic Sulfomucin and sulfate containing proteoglycans are
ionized at pH-1 (low pH): STAIN POSITIVE, rest of sulfomucin and
proteoglycans are negative
 These at pH 2.5 (high pH)

18. Submandibular
salivary gland
mucous cell
showing
sulfomucin
Brown black,
sialomucin blue,
alcian blue
technique
.
REAGENTS:
Alcian Blue Solution:
3% Acetic Acid solution100ml
Alcian blue 8GX -1.0 gm
• Nuclear Fast Red

20. Combined AB and PAS staining
Both technique are combined to differenciate neutral mucins from acidic mucins .

21. Colonic Mucosa showing sialomsucin stain
- magenta High iron diamine +AB stain
Mucicarmine stain
Mucin - deep rose
Nuclei - black
Other tissue elements- yellow

24. Types of
collegen
Location
Type-1 Lung,liver,spleen,kidney,
organic matrix
Type-2 Hyaline and elastic cartilage
Type-3 Found in tissue with type 1 collegen
Type-4 Basement mb
Type-5 Connective tissue cells, endothelial cells, some
epithelial cells

25. Masson's trichrome stain
Van Gieson’s Stain
Verhoeff ’s method (elastic fiber)
Metal impregnation (reticular fiber)

26. 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.

27. A three – dimensional, insoluble protein ‘network’ is formed
(i.e.; protein component of a tissue + fixative in use)
different proteins will form networks with different physical
feature.
For example,
• Erythrocyte protein will produce a very dense network, with
only small pores between the protein elements.
• Muscle cells will form a more open structure with large
pores.
• Collagen will show the least dense network and is quite
porous.

28. .
FACTORS AFFECTING TRICHROME STAINING
• Tissue permeability and dye-molecular size
• Heat (Increases the rate at which staining occurs)
• Acidic pH solution is used (pH- 1.5 to pH 3.0)
Reagents-
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).

29. Human skin
Results
Nuclei – Blue/Black
Cytoplasm, keratin, – Red
muscle fibers,
intercellular fibers
RBCs, Fibrin
Collagen, mucous, – Blue
Cartilage, Amyloid,
adult or mature bone

30. focal segmental glomerulosclerosis (FSGS)
demonstrates blue collagen deposition

31. Van Gieson’s Stain
o Van Gieson’s mixture of picric acid and acid fuchsin
o The simplest method for the differential staining of
collagen.
Principle
 Picric acid provides acidic pH and acts as a
counterstain for muscle and cell cytoplasm.
 It forms a complex with dye which has affinity for
collagen.

32. Nuclei….Black
Collagen.......Brilliant red
Muscle & cytoplasm.....Yellow
Van Giesons Stain

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

34. Verhoff’s Elastic Stain
 Verhoeff's Elastic stain is used to demonstrate pathologic
conditions such as atrophy, breaks, thinning, etc. in elastic
fibers.
principle
 The tissue is over stain with hematoxylin-ferric chloride -
iodine solution.
 Iodine converts disulfide bridges of elastic fibers to anionic
sulphonic acid derivatives. Which react with basic dye
component.

35. IODINE
and
FeCl

36. This section shows elastic cartilage stained with hematoxylin Elastic Fibres – Bluish Black to Black
and potassium iodine to reveal elastic fibers (arrow),
which are dark-stained linear structures embedded
in the cartilage matrix.

37. Demonstration of reticulin fibres
 Reticulin fiber provide supporting framework of cellular organs.
 Reticulin fibres are demonstrated 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.

38. • Permanganate sol
• Change of glycol group to
aldehyde
Tissue
oxidation
• Ferric ammonium sulfate
• Deposition of metallic salt
around the tissue
Sensitization
for metallic
salts
• Ammoniceal Silver solution
• Silver nitrate and Ammonia
Impregnation
with Ag
• Formalin
• Silver is reduced into visible
metallic state
Reduction

39.  The tissue will be oxidize converting the glycol
group to aldehyde followed by a sensitizing step
for metallic salts.
 Impregnation of with silver will follow, then it
will be reduced to a metallic visible state by
formaldehyde.
Gold chloride is applied as a toner which will
change the bound silver from a brown color to a
black color.
A hypo solution will follow in order to remove
any unreduced silver, and if desired a
counterstain is applied

40. Result
o Reticulin fiber – Black
o Collagen – Rose color

41. PIGMENTS AND
MINERALS
ENDOGENOUS
PIGMENTS
HEMATOGENOUS
Iron,
Hemosiderin,
Bile pigments
porphyrins
NON
HEMATOGENOUS
Melanins,
Lipofuscins,
chromaffin,
pseudomela
nosis
ENDOGENOUS
MINERALS
Ca,Cu,
Uric
acid
ARTIFACT
PIGMENTS
Formalin,
malaria Hg,
Schistosome
Chromic
oxide
EXOGENOUS
PIGMENT
AND
MINERALS
Carbon,
silica, Lead,
Silver,
Beryllium
Aluminium

42. Name of pigmemt Colour of
pigment on
H and E
stain
Special stain of
pigment
Colour of
pigment on stain
Iron Brown Pearls prussian blue Blue
Bile Yellow-brown Foutchet’s Blue to green
Melanin,lipofuscins,
chromaffin
Brown-black -Masson-fontana
-Schmorl’s reaction
Black
Dark blue
Lipofuscins Brown Long ziehl neelson Megenta
Calcium Purple -blue Von kossa’s silver nitrate Black
copper Colourless to
brown
Rhodanine technique Red- orange
Uric acid, urates colourless Li carbonate extraction-
hexamine silver
Black
silver colourless Rhodanine method Reddish -brown

43. Bleach, DOPA
Pos: melanin Neg; lipo (Long ZE)
negative
lipofuscin melanin
Lipofuscin, melanin, chromaffin
Chromaffin stain

44. PERL’S- PRUSSIAN BLUE
REACTION
PRINCIPLE :
The reaction occurs with the treatment of sections in acid solutions of
ferrocyanides. Any ferric ion in the tissue combines with the ferrocyanide
and results in the formation of a bright blue pigment called 'Prussian
blue" or ferric ferrocyanide.
PURPOSE:
o To demonstrate ferric iron in tissue sections.
o Small amounts of iron are found normally in spleen and bone marrow.
o Excessive amount are present in hemochromatosis , hemosiderosis, with
deposits in the liver, spleen, and lymph nodes.

45. Hemosiderin, liver, iron stain.
RESULTS:
Iron (hemosiderin) -blue
Nuclei -red
Background - pink
REAGENTS:
Ferrocyanide solution
•1% aqueous Potassium Ferrocyanide:
•2% aqueous Hydrochloric Acid:
Nuclear-fast Red:

46. PURPOSE:
 To identify argentaffin granules, lipofuscins and melanin .
 Melanin is a nonlipid, non hematogenous pigment.It is a brown-black
pigment present normally in the hair, skin, retina, iris and certain parts of
the CNS.
 Argentaffin granules are found in carcinoid tumors
PRINCIPLE:
 The solutions of ammoniacal silver nitrate are reduced by melanin to
black metallic silver this is the basis of Massons Fontana method for
demonstrating melanin.
.

47. Melanin pigment of skin showing black color
Melanin -black
Nuclei -red
Melanin pigment in cells of malignant melanoma,
Fontana-Masson stain.
REAGENTS:
oAmmoniacal Silver Solution:
• 10% Silver nitrate 20 ml
• Ammonia.
oNuclear-Fast Red:

48.  Bleaching techniques use strong oxidizing agent to remove
melanin to get a good cellular morphology.
 Method of choice is peracetic acid, but 0.25% potassium
permanganate followed by 2% oxalic acid also work well.
 Other oxidizing agents,such as chlorate, chromic acid,
peroxide also bleach melanin.
 Formalin pigment-It is removed from tissue section by
pretreatment with saturated alcoholic picric acid solution.
 Mercury- treatment of section with lugol’s iodine is classical
method of removing this pigment

50. Coronary artery showing calcified atheromatous
plaque
von kossa staining
Calcium salts -black
Nuclei -red
Cytoplasm -pink
Hall’s method
Bile-Green

51.  Copper deposition seen in
Wilson’s disease and chronic
biliary diseases.
Stain-- Rhodanine (choice)
- Rubeanic acid method
- Orcein
 Rhodanine stain shows bright
orange to brown granules of Cu
within hepatocytes and nuclei in
blue.

52. STAINS FOR LIPID
- Oil Red O
- Sudan Black B
 Purpose-
 To demonstrate fat or lipids in fresh tissue sections.
 Fat occurring in an abnormal place, such as fatty emboli
 Tumors arising from fat cells (liposarcomas) can be
differentiated from other types of tumors.
 Principle-Fat dissolved in processing, demonstrated
in Formal calcium, NS: Frozen sectioning.
 Staining with oil-soluble dyes is based on the greater
solubility of the dye in the lipid substances .

53. SUDAN BLACK B STAINING
PRINCIPLE
 Sudan Black is slightly basic dye and will combine with acidic
groups in compound lipids.
 thus staining phospholipids also.
PURPOSE :
 For the demonstration of fat.
REAGENTS :
 85% Propylene Glycol:
 Hematoxylin:
 Sudan Black B

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