FIXATION: 10% buffered neutral formalin. Fix smears in absolute methyl alcohol.
TECHNIQUE: Paraffin or plastic sections cut at 5 µm. Smears and touch preparations may be used.
SOLUTIONS:
Phosphate Buffer Solution pH 7.6
Sodium phosphate, dibasic, Na2 HPO4 ---------------------------------------- 3.5 gm
Potassium phosphate, monobasic, KH2 PO4 ----------------------------------- 0.5 gm
Distilled water to make a total of --------------------------------------------- 400.0 ml
Alpha-Naphthyl Acetate Solution
Alpha-naphthyl acetate -------------------------------------------------------- 0.04 gm
Ethylene glycol monomethyl ether --------------------------------------------- 2.00 ml
0.3% Sodium Borate
(see PAS Method)
1. Deparaffinize and hydrate sections to distilled water.
2. Mix 1.2 ml of 4% hexazotized pararosanilin and 1.2 ml of 4% sodium nitrite in a 50 ml flask and let stand for one minute. To this add 36 ml of phosphate buffer pH 7.6, mix and add to the alpha-naphthyl acetate solution and mix well. Adjust the pH to 6.1 with 1N hydrochloric acid (range 5.8-6.5 is acceptable). Filter and immediately place slides in this solution and stain for 45 minutes.
3. Wash slides in running tap water for 5 minutes.
4. Rinse in distilled water.
5. Counterstain with acidified Lillie-Mayer or Harris hematoxylin for 1 minute.
6. Rinse in three changes of distilled water.
7. Blue hematoxylin in 0.3% sodium borate for 15 seconds.
8. Rinse in four changes of distilled water.
9. Air dry the slides.
10. Dip in xylene and mount with synthetic resin.
Nonspecific esterase in the cytoplasm of monocytes,
Histiocytes and megakaryocytes -------------------------------------- brown-red
Nuclei ---------------------------------------------------------------- blue
Brown, G.G.: An Introduction to Histotechnology, New York, Appleton-Century-Crofts, 1978, pp. 346-347.
Yam, L.T., Li, C.Y., and Crosby, W.H.: Cytochemical identification of monocytes and granulocytes. Am. J. Clin. Pathol. 55:283-290, 1971.
Image Examples:
Diagnostic Application: