Sudan IV Stain, Herxheimer Alcoholic

SOLUTION:

Additionally Needed:

For storage requirements and expiration date refer to individual bottle labels.

APPLICATION:

Newcomer Supply Sudan IV Stain, Herxheimer Alcoholic procedure is used for identification of fat/lipid in frozen sections. Herxheimer method refers to an acetone/alcohol solvent mixture; the acetone component of this solution may dissolve out small amounts of lipid.

Sudan dyes are a group of fat/lipid soluble solvent dyes, also known as lysochromes.  These solvent dyes readily stain fat/lipid since the dyes are more soluble in lipid than in the solvents from which they are applied.

METHOD:

Fixation: Fresh tissue or formalin fixed unprocessed tissue

1. 1. 1. See Procedure Notes #1 and #2.

Technique: Frozen sections cut at 8 microns on adhesive slides

Solutions:  All solutions are manufactured by Newcomer Supply, Inc.

All Newcomer Supply stain procedures are designed to be used with Coplin jars filled to 40 ml following the provided staining procedure.

STAINING PROCEDURE:

1. 1. Fix frozen section slides in Formalin 10%, Phosphate Buffered (Part 1090) for 1 minute.
   2. Rinse sections in two changes of distilled water.
   3. Rinse in Alcohol, Ethyl Denatured, 70% (Part 10844).
   4. Stain in Sudan IV Stain, Herxheimer Alcoholic for 10 minutes.
      1. 1. Keep tightly capped to avoid evaporation.
   5. Differentiate quickly in Alcohol, Ethyl Denatured, 70%.
   6. Wash thoroughly in distilled water.
   7. Stain with Hematoxylin Stain, Mayer Modified (Part 1202); 2-3 minutes.
   8. Wash in several changes of tap water.
   9. Optional: Blue in Lithium Carbonate, Saturated Aqueous (Part 12215) or Scott Tap Water Substitute (Part 1380) for 10 dips.
   10. Wash in several changes of tap water.
   11. Blot excess water from slide; coverslip with Mount-Quick Aqueous (Part 6271A) mounting medium.
       1. 1. See Procedure Notes #3 and #4.

RESULTS:

PROCEDURE NOTES:

1. 1. Freeze tissue according to laboratory protocol.
   2. To freeze formalin fixed tissue that has not been processed:
      1. 1. Wash tissue in running tap water for 5 minutes.
         2. Blot excess water from tissue and freeze.
   3. Use minimal pressure with coverslip application to avoid displacement of fat/lipid staining.
   4. To remove trapped air bubbles or to recoverslip;
      1. 1. Soak slide in warm water until coverslip falls off.
         2. Blot excess water from slide.
         3. Remount with new coverslip and Mount-Quick Aqueous.

REFERENCES:

1. 1. Culling, C. F. A. Handbook of Histopathological and Histochemical Techniques: (including Museum Techniques). 3rd ed. London: Butterworth, 1974. 359-362.
   2. Kiernan, J. A. Histological and Histochemical Methods: Theory and Practice. 3rd ed. London, Ontario: Arnold, 2003. 251-254.
   3. Lillie, R. D. and Harold Fullmer. Histopathologic Technic and Practical Histochemistry. 4th ed. New York: McGraw-Hill, 1976. 565-568.
   4. Modifications developed by Newcomer Supply Laboratory.