A new colorant for megakaryocytes.

Intended Use :

Megacolor is intended for use as a selective colorant for the cytoplasm of megakaryocytes, and consequently as a way of identifying these cells.

Principle :

MEGACOLOR selectively and metachromatically stains the cytoplasm of mature and immature megakaryocytes in specimens of blood and bone marrow that have been pretreated with ribonuclease to remove RNA. Used in conjunction with immunoperoxidase and immunocytochemical techniques, staining of megakaryocytes with MEGACOLOR may represent a rapid screening technique for these cells.

Reagents :

1. Absolute methanol.
2. Sodium acetate.
3. Magnesium sulfate.
4. Ribonuclease A (bovine pancreas type III A, Sigma Catalog number R 5125).
5. MEGACOLOR stain.

Procedure :

1. Fix coverslips containing peripheral blood, buffy coat, or bone marrow in absolute methanol for 5 minutes. Wash and air dry.
2. Just before use, prepare sodium acetate 1.4 grams/10 mL distilled water and magnesium sulfate 2.46 grams/10 mL distilled water. Combine these two solutions and add 80 mL distilled water. Dissolve 4 mg ribonuclease A in 50 mL of the latter solution.
3. Incubate coverslips in ribonuclease solution for 5-10 minutes at 37 degrees C. Wash in distilled water and air dry.
4. Stain the ribonuclease treated coverslips with MEGACOLOR for 10 minutes. The presence of undissolved dye particles in the stain solution does not affect the performance of the stain.
5. Grasp the coverslip with a forceps, drain off excess stain onto a piece of filter paper and agitate vigorously in a beaker containing distilled water for 10 seconds.
6. Blot using filter paper, and mount with synthetic xylene based resin mounting medium on cleaned, labeled glass slides for light microscopy.

Results :

In mature and immature megakaryocytes viewed with a didymium filter, the cytoplasm stains intense magenta, a color not found in other types of blood and bone marrow cells. In eosinophils, granules stain pink. In other types of cells, the cytoplasm stains pale yellow to yellow brown, except in megakaryocytes, where the nuclei stain brown.