special stains in histopathology

1. SPECIAL STAINS
IN
HISTOPATHOLOGY

2. ● H and E stain
– Preliminary stain ( first stain) applied to the tissue
sections.
– Provides diagnostic information in most of the
cases.

3. ● Special stain
– Staining technique that is used to highlight various
individual tissue components which can not be
identified in routine H and E stain or are better
appreciated on special stains.
– Eg. special stains for carbohydrates, amyloids,
nucleic acids, lipids, microorganisms etc.

4. Special Stains for Lipids

5. ● LIPIDS
● Simple lipids
– Fat
– Oil
– waxes
● Compound lipid
– C/o fatty acids,
alcohol and
another group
eg. phosphorus
or nitrogen
● Derived lipids
– Derived from simple or
compound lipids by
hydrolysis
– Cholesterol
– Bile acids

6. ● Lipids with melting points below staining
temperature can be stained with fat stains.
● Lipids in solid or crystalline form at staining
temp. can not be stained.
● Melting point is inversely related to the length of
fatty acid.
● Best fixatives- Formal calcium (2% calcium
acetate and 10% formalin)

7. ● Staining techniques for lipids
– Oil red O
– Sudan black B

8. Oil Red O
● Principle
– Staining with oil soluble dyes is based on greater
solubility of the dye in lipid substances than in usual
hydroalcoholic dye solvents.
● Result
Lipid – RED
Nucleus – blue

9. Reagents
● 60% isopropanol
● Alum hematoxylin
● Glycerin jelly mounting medium
● Red oil O working solution.

10. ● To make oil red O stock solution
– Oil Red O 0.5g
– Isopropanol 100 ml
dissolve the dye in isopropanol, using very gentle
heat of water bath. This is the stock solution.
● To make working oil red O solution
– Dilute 30ml of stock solution in 20ml of distilled
water and allow it to stand for 10 min.
– Filter into coplin jar and cover immediately.
– Working solution should be prepared fresh each
time.

11. Procedure
● Fix slides in 10% formalin if fresh.
● Wash well in tap water for 1 – 10 min to drain of
excess water.
● Rinse with 60% isopropanol.
● Stain with freshly prepared Red oil O working
solution for 15 min.

12. ● Dip in alum hematoxylin (5 dips) – stains
nucleus.
● Rinse with distilled water
● with aqueous mounting media, Glycerine Jelly.

13. ● Fat emboli in Oil red O stain.

14. Uses of Oil Red O stain
● Oil red O stain is used for staining neutral
triglycerides, lipids and lipoprotein.
● Tumors arising from fat cells (liposarcomas)
can be differentiated from other types of
tumors.
● Fat occurring in an abnormal place, such as
fatty emboli that may develop after either a
bone fracture or an injury that crushes a fatty
body area.

15. ● Lipid storage disorder like nieman-pick disease
● To demonstrate fat or lipids in condition like
fatty liver.
● In hematological condition like burkitt’s
lymphoma and sea-blue histiocytic syndrome.

16. Sudan Black B
● Principle
– Sudan Black B is a dye insoluble in water but
dissolves in fat. Therefore the dye accumulates in
fat globules within the cells.
– It is a slightly basic dye and combines with acidic
groups in compound lipids, thus staining
phospholipids too.
● Results
– Fats : blue/black
– Nucleus : red

17. ● Reagents
– 85% propylene glycol
Propylene glycol : 85 ml
Distilled water : 15 ml
– Hematoxylin
– Sudan black B/ Propylene
Sudan Black B : 0.7 g
Propylene glycol : 100ml

18. ● Procedure
– Fix slides in 10% formalin.
– Wash well in tap water, rinse in distilled water, and
drain off excess water.
– Dip in propylene glycol – 2 changes – five minutes
each (dehydration)
– Stain with sudan black B – 7 minutes
– Dip in 85% propylene glycol – 3 minutes.
(differentiation)

19. ● Rinse in distilled water.
● Stain with nuclear fast red – 3 minutes.
● Wash in tap water
● Rinse in distilled water.
● Mount using aqueous media.

20. ● Sudan black B - myeloblasts

21. Uses of Sudan Black B
● Used for staining neutral triglycerides, lipids,
lipoproteins and phospholipids.
● In hematological disorders, it can be used to
differentiate blasts. Sudan black B stains
myeloblasts but does not stain lymphoblasts.

22. Thank You