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SUDAN BLACK B FOR PHOSPHOLIPIDS IN PARAFFIN SECTIONS
AND FATS IN FROZEN SECTIONS
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FIXATION: 10% buffered neutral formalin.
TECHNIQUE: Paraffin sections cut at 5 μm or frozen sections cut at 8 μm.
SOLUTIONS:
Sudan Black B Solution
Sudan black B, C.I. 26150 ------------------------------------------------------ 1.0 gm
Isopropyl alcohol ----------------------------------------------------------------- 70.0 ml
Place on a magnetic stirrer and allow to mix for 30 minutes.
Dissolve 0.3 gm of dextrin in 30 ml of distilled water and add to
the Sudan black B solution. Mix for 5 minutes and filter through
Whatman #4 filter paper into an amber bottle. Filter again before use.
0.1% Nuclear Fast Red Solution
(see Pearls Method for Iron)
Glycerin Jelly
Gelatin --------------------------------------------------------------------------- 10.0 gm
Distilled water -------------------------------------------------------------------- 60.0 ml
Heat until gelatin is dissolved. Add:
Glycerin ------------------------------------------------------------------------- 70.0 ml
Phenol, melted ------------------------------------------------------------------- 1.0 ml
1. Deparaffinize and hydrate to 70% alcohol. When staining frozen sections, begin at step 3.
2. Stand slides on end and allow to dry.
3. Place in Sudan black B solution for one to three hours. Check after one hour but first dip in 70% isopropyl alcohol. When staining frozen sections, stain for 20 minutes at room temperature.
4. Rinse thoroughly in two changes of 70% isopropyl alcohol.
5. Wash with six changes of distilled water.
6. Nuclear fast red solution for 3 minutes.
7. Rinse in two changes of distilled water.
8. Mount with glycerin jelly.
Phospholipids ------------------------------------------------------- black or blue-black
Fats ----------------------------------------------------------------- black or blue-black
Erythrocytes --------------------------------------------------------- blue-black
Nuclei --------------------------------------------------------------- pink
When paraffin sections are stained with this procedure only undissolved structural lipids (phospholipids) are stained. Fats present in frozen sections stain intensely black or blue-black.
The addition of dextrin to the Sudan black B helps to stabilize the solution and thus extend its shelf-life (six months or longer).
Disbrey, B.D., and Rack, H.H.: Histological Laboratory Methods, E. and S. Livingston, London, 1970, 162-163.
Lison, L. and Dagnelie, J.: Methods for the Staining of Myelin. Bull. Histol. Appl. Physiol. Path. 12:85, 1935.
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