Prepared by

ROY ELLIS

IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011

Principle

By matching the ionic strength and/or the pH of the solvent, solutions of the copper phthalocyanins (eg Alcian blue) are used to react with (and thus demonstrate), the ionisable moiety of acidic proteoglycans (mucins).  The moiety in question can be:

sulphated residue

carboxylic residue

hyaluronic residue

chondroitin residue

Technical Points

1.  (step 4) Red background staining results if the neutral red stain is applied directly from tap water.

2.   Controls section as required.

            For Alcian blue pH 2.5 use section of colon

            For Alcian blue pH 1.0 or 0.2 use section of cartilage

Method

1. Bring sections to distilled water
2. Stain in the Alcian blue solution 15 mins
3. Wash well in running tap water 5 mins (For lower pH solutions ie pH 1.0/0.2, drain and blot dry, to prevent removal of stain in water).
4. Rinse in distilled water
5. Counterstain with neutral red stain 1 min
6. Rapidly dehydrate in absolute alcohol, clear and mount.

Results

• acid mucins……………………………… blue
• nuclei………………………………………… red
• erythrocytes……………………………… yellow

At pH 2.5 most acid mucins (except some of the strongly sulphated groups)........... Blue

At pH 1.0 only weakly and strongly sulphated acid mucins .................... Blue

At pH 0.2 only strongly sulphated acid mucins ....................Blue

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Reagent Formulae

1.  Alcian Blue pH 2.5

  1% Alcian blue in 3% aq acetic acid

             Alcian blue 8GX (CI 74240) ------ 1.0 g

             distilled water -------------------- 97.0 ml
             glacial acetic acid ---------------- 3.0 ml
Dissolve the dye in the distilled water, add acid, mix well.  Filter into the reagent bottle and filter before use.

2. Alcian blue pH 1.0

   Dissolve 1 gm of alcian blue in 90mls of distilled water and 10mls of 1N hydrochloric acid.

                (Hydrochloric acid 1N     Distilled water ........................ 915 ml)

              Concentrated hydrochloric acid ..... 85 ml

3.  Alcian blue pH 0.2:

Dissolve 1 gm alcian blue in 100mls of 10% sulphuric acid.

(Sulphuric acid 10%  Distilled water .......................... 90ml)

Concentrated sulphuric acid.......... 10ml)

4. Neutral red stain
      neutral red (CI 50040) ----------- 1.0 g
      distilled water -------------------- 100 ml
      gl acetic acid ---------------------- 0.1 ml
Dissolve the dye in the distilled water.  Add the acid and mix well.  Filter into the reagent bottle (keeps well).

References

1.    Bancroft, J.D. and Stevens, A. 1982Theory and Practice of Histological Techniques. 2nd edition.Churchill-Livingstone.

2.    Lillie, R.D. 1977. H.J. Conn's Biological Stains, 9th edition Williams and Wilkins Pearse, A.G. Everson 1972

3.    Histochemistry, Theoretical and Applied Vol. 1, 3rd edition, p344 Churchill-Livingstone.

4.    Steedman, H.F. 1950 Alcian blue 8GS; A new stain for mucin. Quarterly Journal of Microscopic Science, Vol 91, p477-479.

5.    Ajax Chemicals 1982-89Material Safety Data Sheets Ajax Chemicals Pty Ltd, Auburn, New South Wales, Australia.

6.    National Society for Histotechnology, Carbohydrate Reference Stain Manual, 1^st edition, 1989.