Histotechnology Technical methods

Enzyme Histochemical Stain

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Unfixed cryostat or cold Formol calcuim/gum sucrose fixed on muscle biopsy sections.


a. 0.1M Glycine buffer.

  • 0.75g glycine + 0.585g NaC1 made up to 100ml with distilled water.

b. O.1M glycine/NaC1 buffer with 0.75M CaC12

  • 50ml 0.1M glycine buffer
  • 10ml 0.75M CaC12 -11.03g CaC12 2.H20/100ml.
  • Add approx. 22ml 0.1M NaOH until 9.6-9.8 pH

METHOD At pH 9.4.

  1. Incubate freshly cut sections at 37C for 11 minutes, in the following
  2. solution
    • 5mg ATP. dissolved. Add 10ml of solution (b) adjust to pH 9.6-9.8.

  3. Rinse well in distilled water.
  4. Immerse in 2% COC12 for 5 minutes.
  5. Rinse well in distilled water + 3 or 4 in tap water.
  6. Immerse in dilute (1:10) ammonium sulphide solution for 30 seconds.
  7. Rinse well in running tap water.
  8. (optional) stain in Harris's haematoxylin, blue in tap water.
  9. Mount in glycerine jelly.

METHOD At pH 4.2 and 4.6

  1. Preincubate at 4C in 0.1M sodium acetate buffer with 10mM ETDA added (0.372g/100ml of buffer) for 10 minutes at pH 4.2 or 4.6.
  2. Wash in distilled water.
  3. Proceed as for the pH 9.4 method.


Only the pH 4.2 sections need counterstaining in haematoxylin. Sections must be VERY well washed after steps 3 and 5 to remove any traces of previous solutions

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