Burstone's Staining Protocol

for Alkaline Phosphatase on Frozen Tissue Sections


NovaUltra Special Stain Kits


Solutions and Reagents:


Naphthol AS-MX Phosphate/(DMF)N,N Dimethylformamide Solution:

      Naphthol AS-MX Phosphate ----------------- 5 mg

      N,N Dimethylformamide -------------------- 0.25 ml

      Mix to dissolve.


Tris-hydrochloric acid buffer, pH 8.74:

      0.2 M Tris (2.42 g Tris Base/100 ml) ----- 10 ml

      0.1 M Hydrochloric acid (HCl) --------------- 4 ml

      Distilled water -------------------------------- 26 ml


Substrate Working Solution:

      Naphthol AS-MX phosphate/DMF solution ----- 0.25 ml

      Distilled water ------------------------------------ 25 ml

      Tris buffer, pH 8.74 ----------------------------- 25 ml

Shake to mix thoroughly and then add 30 mg of diazonium salt (fast red violet LB salt). Filter:

Insure that the ingredients are thoroughly mixed before filtering mixture into a oplin jar.

Solution should be clear (Solution maybe slightly cloudy which should be OK for the staining)

and canary yellow (Once the solution turns reddish, it will not stain sections)


Mayer’s Hematoxylin Solution




1.   Cut fresh frozen sections and mount on Superfrost Plus slides. Fix in ice cool acetone for 5 minutes. Allow sections air dry for about 5 minutes. Place sections into substrate working solution at 37 C for 30-60 minutes. Check slides microscopically after 30 minutes for the intense red color indicative of enzyme activity (the background should be yellow).

2.   Rinse in distilled water. 3x2 minutes.

3.   Counterstain, if desired, Mayer’s hematoxylin for 1-2 minutes (For capillary staining, DON’T counterstain for it will be very difficult to distinguish between capillary and nucleus).

4.   Rinse in distilled water.

5.   Mount DIRECTLY with aqueous medium, since alcohol and xylene will ERASE the staining.




      Sites of enzyme activity -------------------- pink to red

      Nuclei ----------------------------------------- blue  

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Positive Controls:


      Smooth muscle (intestine or colon), heart muscle.