Histotechnology Technical methods

Connective Tissue Stain

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4µm paraffin wax sections



  • Haematoxylin solution
    • Haematoxylin 1g
    • Absolute alcohol 100mls

    Allow to ripen naturally for 4 weeks before use

  • Iron solution
    • 30% aqueous ferric chloride
    • Concentrated hydrochloric acid 1ml
    • Distilled water 95mls

This solution is filtered and added to an equal volume of the haematoxylin solution immediately before use. The mixture should be a violet/black colour and must be discarded if it is brown.


  • Equal volume s of 0.5% ponceau 2 R in 1% acetic acid and 0.5% acid fuchsin in 1% acetic acid.

3. 1% phosphomolybdic acid

4. 2% light green in 2% citric acid diluted 1:10 with distilled water prior to use

5. Aniline Blue

  • Boil 97.5mls distilled water and add 2g Aniline Blue while still hot. Then add 2.5mls glacial acetic acid, cool and filter


  1. Take sections to water
  2. Stain nuclei with Weigerts iron haematoxylin for 10 minutes
  3. Wash in water and 'blue' in lithium carbonate
  4. Stain in 1% panceau-acetic acid solution for 5 minutes
  5. Rinse rapidly in water
  6. Differentiate in 1% phosphomolybdic acid for approximately 5 minutes
  7. Drain and counterstain with light green or aniline blue
  8. Dehydrate, clear . Mount sections in DPX


  • Nuclei - Blue/black
  • Muscle, red blood cells, fibrin - Red
  • Connective tissue - Blue or green

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