Stain: Acid Phosphatase

More Staining Procedures For Plastic Embedded Tissue


To demonstrate acid phosphatase activity


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


  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


Acid phosphatase activity- vivid red
Nuclei- blue


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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