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Enzyme Histochemical Stain

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5-10, micron unfixed, or cold formol calcium/gum sucrose fixed Cryostat sections.

Stock solutions
A) Pararosanilin-HCL Stock

  • Pararosanilin 1g
  • Distilled water 20ml
  • HCl Conc 5ml
The pararosanilin is dissolved in the distilled water and the hydrochloric acid is added. The solution is heated gently, cooled, filtered and stored in aliquots in a refrigerator.

B) Sodium Nitrite

  • Sodium nitrite 2g
  • Distilled water 50ml

This solution has to be prepared Fresh or made into 0.4ml aliquots and stored in the deep freeze.

C) Veronal-Acetate Buffer Stock

  • Sodium Acetate (3H20) 3.88g
  • Sodium Barbitone 5.88g
  • Distilled water 200ml

D) Naphthol ASB1 Phosphate Stock

  • Naphthol ASB1 Phosphate 50mg
  • Dimethyl formamide 5ml

This is put into 0.5ml aliquots and stored in the deep freeze.

Preparation of incubating solution

  • Pararosanilin - HCL (A) 0.4ml
  • Sodium nitrite (40mg/ml) (B) 0.4ml

Add the pararosanilin drop by drop to the thawed sodium nitrite, shaking well after each addition until the solution is corn-coloured (leave to stand for at least 30 seconds).

  • Naphthol ASB1 phosphate (D) 0.5ml
  • Veronal Acetate Buffer Stock (C) 2.5ml
  • Distilled water 6.5ml

Mix these together well and then add the pararosanilin/sodium nitrite solution. Adjust the pH to 4.7-5.0, filter and use immediately.


  1. Incubate sections for 10-60 minutes at 37C
  2. Wash well in distilled and then tap water
  3. Counterstain, with 2% Methyl green for 15-30 seconds (haematoxylin can be used as an alternative)
  4. Wash
  5. Dehydrate, clear . Mount sections in DPX


  • Acid phosphatase activity - Red


It is important to wash the sections well after the incubation as the acidity of the solution can make the haematoxylin staining muddy. If at any point the incubating solution is bright red the method must be repeated as this means that the sodium nitrite and pararosanilin have not reacted adequately. The incubating medium should be a slightly opalescent straw colour.

For demonstrating tartrate resistent acid phosphatase add 28mg/10ml of sodium tartrate to the incubating solution. Remember to use a control batch without sodium tartrate using test and control sections.

NOTE: No responsibility is assumed by The University of Nottingham or the Queens Medical Centre NHS Trust for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. It is the users responsibilty to ensure that all procedures are carried out according to appropriate Health and Safety requirements.

© Copyright 1997 University of Nottingham Medical School Division of Histopathology. This page was last built on Mon, May 5, 1997 with Frontier. Thanks for looking in. Comments to James.Lowe@nottingham.ac.uk