Stain: Acid Phosphatase

More Staining Procedures For Plastic Embedded Tissue

INDICATION:

To demonstrate acid phosphatase activity

SOLUTIONS:

Michaelis Vernonal Acetate buffer pH 5.0
5 ml stock veronal acetate
1.94 g sodium acetate trihydrate
2.94 g sodium barbitone
100 ml distilled water
9 ml 0.1 M hydrochloric acid
0.85 ml concentrated hydrochloric acid
100 ml distilled water
9 ml distilled water

Substrate solution (MAKE FRESH):
100 mg Naphthol AS-TR (Sigma N5875)
10 ml N,N dimethylformamide (Sigma D4254)

4% pararosaniline:
2 g pararosaniline HCl (Sigma P3750), CI 42500)
50 ml 2N HCl

4% sodium nitrite (MAKE FRESH):
0.4 g sodium nitrite
1 ml distilled water

Hexaxotized pararosaniline (MAKE FRESH)
0.8 ml pararosaniline solution
0.8 ml sodium nitrite
Mix for 1 minute

Working substrate (MAKE FRESH):
5 ml Michaelis Veronal-Acetate buffer
12 ml distilled water
1 ml substrate
1.6 ml hexazotized pararosaniline
Adjust pH to 5.0 with NaOH. Filter

Gill's hematoxylin

PROCEDURE:

  1. Incubate sections in working substrate for two hours at room temperature
  2. Rinse with distilled water
  3. Counterstain with Gill's hematoxylin 1 to 2 minutes
  4. Rinse well with tap water
  5. Dry and mount

RESULTS:

Acid phosphatase activity- vivid red
Nuclei- blue

REFERENCE:

Brinn, NT and Pickett, JP., "Glycol Methacrylate Routine, Special Stains, Histochemistry, Enzyme Histochemistry and Immunohistochemistry- A Simplified Cold Method for Surgical Biopsy Tissue," J of Histotechnology 2(3):125-130, 1979


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