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NAPHTHOL AS - D CHLOROACETATE METHOD FOR
ESTERASE IN PARAFFIN SECTIONS AND SMEARS
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FIXATION: 10% buffered neutral formalin. Avoid fixative containing mercury. For smears and touch preparations fix for two minutes in absolute methanol containing 10% formaldehyde.
TECHNIQUE: Paraffin or plastic sections cut at 5 µm. Smears and touch preparations may be used.
SOLUTIONS:
4% Hexazolized Pararosanilin
Pararosanilin, C.I. 42500 ------------------------------------------------------- 1.0 gm
Distilled water ------------------------------------------------------------------ 20.0 ml
Hydrochloric acid, concentrated ------------------------------------------------ 5.0 ml
4% Sodium Nitrite
Sodium nitrite ------------------------------------------------------------------- 0.4 gm
Distilled water ------------------------------------------------------------------- 10.0 ml
Phosphate Buffer, pH 6.5
Sodium phosphate, dibasic, Na2 HPO4 ---------------------------------------- 0.652 gm
Potassium phosphate, monobasic, KH2PO4 ------------------------------------ 1.348 gm
Distilled water ------------------------------------------------------------------ 200.000 ml
Naphthol AS - D Chloroacetate Solution
Naphthol AS - D chloroacetate ------------------------------------------------ 0.01 gm
N,N – dimethylformamide ----------------------------------------------------- 1.00 ml
Prepare shortly before use.
0.3% Sodium Borate
(see PAS Method)
1. Deparaffinize and hydrate sections to distilled water.
2. Mix one drop of 4% p-rosanilin in 2N hydrochloric acid and one drop of 4% sodium nitrite in a 50 ml flask and let stand for one minute. To this add 40 ml of phosphate buffer pH 6.5 and mix. Add this solution to the naphthol AS - D chloroacetate solution, mix well, and filter. Immediately place slides in this solution and stain for 45 minutes.
3. Rinse slides in four changes of distilled water.
4. Counterstain with acidified Lillie-Mayer hematoxylin for 1 minute.
5. Rinse in three changes of distilled water.
6. Blue hematoxylin in 0.3% sodium borate for 15 seconds.
7. Rinse in four changes of distilled water.
8. Air dry the slides
9. Dip in xylene and mount with synthetic resin.
Leukocyte esterase --------------------------------------------------- red
Mast cells ------------------------------------------------------------ red
Nuclei ---------------------------------------------------------------- blue
The staining of esterase is inhibited to varying degrees by mercury, acid solutions, heat and iodine. Poor or false negative results occur when:
1. Slides are overheated when drying.
2. Mercury bichloride crystals are removed from tissue with an iodine solution.
3. Tissues are fixed in an acid fixative such as Zenker-formalin, Zenkers with acetic acid or Bouins fluid.
Brown, G.G.: An Introduction to Histotechnology, New York, Appleton-Century-Crofts, 1978, pp. 345-346.
Leder, L.D.: The selective enzymocytochemical demonstration of neutrophil myeloid cells and tissue mast cells in paraffin sections. Klinische Wochenchrift, 42:553, 1964.
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