Histotechnology Technical methods

Stain for sulphomucins

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8µm paraffin wax sections


1. High iron diamine:

  • N, N-dimethyl-meta-phenylenediamine dihydrochloride 120 mg
  • N, N-dimethyl-para-phenylenediamine dihydrochloride 20 mg
  • Distilled water: 50 ml
  • Ferric Chloride (60% BDH solution) 1.4 ml

Dissolve the two diamine salts simultaneously in the distilled water, then add to the ferric chloride solution and mix. (The mixing is best done in a Coplin jar.)

2. 1% alcian blue in 3% acetic acid

3. 0.5% aqueous neutral red


  1. Take positive control and the test sections to distilled water.
  2. Place sections in the high iron diamine solution for 18-24 hours at room temperature.
  3. Wash well in running water.
  4. Stain with the alcian blue solution for 5 minutes.
  5. Wash in water, stain nuclei with neutral red solution for 2-3 minutes.
  6. Wash in water. Dehydrate, clear . Mount sections in DPX


  • Sulphated mucins: black/brown
  • Carboxylated mucins: blue
  • Nuclei: red


  • If the times of incubation are exceeded then non-sulphated mucins become stained and the method loses specificity.

  • Ferric chloride is used as a 60% stock solution which should be no older than 2 weeks.

  • A heavy background staining is evidence of a deteriorated ferric chloride solution or diamine. The shelf-life of diamine salt (dry) is about one year.

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