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Luna Staining Method and Protocol for Elastic Fibers and Mast Cells
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Luna Staining Method and Protocol for Elastic Fibers and Mast Cells

Description: This method is used for identifying elastic fibers and mast cells on formalin-fixed, paraffin-embedded tissue sections, and may be used for frozen sections as well. The elastic fibers and mast cells will be stained purple and the nuclei will be stained black and the background stained yellow. 

Fixation: 10% formalin.

Section: paraffin sections at 5 um.

Solutions and Reagents:

Aldehyde Fuchsin Solution:

      Basic fuchsin ---------------------------- 1 g

      70% Ethyl alcohol ----------------------- 200 ml

      Hydrochloric acid, concentrated ------ 2 ml

      Paraldehyde ----------------------------- 2 ml

      Let stand for 2-3 days. Filter just before use.  Store solution at 4 C and it will be good for a few weeks.

Iron Hematoxylin Working Solution, Weigert's:

      Stock solution A: 1 g hematoxylin in 100 ml 95% alcohol.

      Stock solution B: 4ml 29% ferric chloride in 95 ml distilled water, then add 1 ml concentrated hydrochloric acid.

      Working solution: mix equal parts of stock solution A and B. This working solution is reusable within 3 months.

Methyl Orange Solution:

      Methyl orange (Sigma) ---------------- 0.25 g

      Alcohol (95%) ---------------------------- 100 ml

      Mix well and filter before use.

Procedure:

1.   Deparaffinize and hydrate sections to 95% alcohol.

2.   Stain sections in aldehyde fuchsin solution for 30 minutes.

3.   Rinse in 95% alcohol, 3 changes (for differentiation as well).

4.   Stain in Weigert’s iron hematoxylin working solution for 10 minute.

5.   Wash in running tap water for 10 minutes.

6.   Rinse in 95% alcohol.

7.   Counterstain in methyl orange solution for 5 minutes.

8.   Dehydrate through 95% alcohol, absolute alcohol for 2 changes, 3 minutes each.

9.   Clear in xylene, 2 changes, 3 minutes each.

10. Coverslip with resinous mounting medium.

Results:

      Elastic fibers -------------------------- purple

      Mast cells ------------------------------ purple

      Nuclei ------------------------------------ black

      Background ---------------------------- yellow 

Positive Controls:

      Skin.

References:

1) Lambert C, and Futch HN (1976) An evaluation of elastic tissue staining. Am J Med Technol 42:305-309.

2) Luna L. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. New York, NY: McGraw-Hill; 1968:114-115.

3) Sheehan D, Hrapchak B, Theory and Practice of Histotechnology, 2nd Ed, 1980, pp282, Battelle Press, Ohio.

4) Prophet EB, Mills B, Arrington JB, Sobin LH: Laboratory Methods in Histotechnology. pp145. Washington, DC: American Registry of Pathology, 1992.

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