FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin or plastic sections cut at 3-4 μm.

SOLUTIONS:

3% Acetic Acid Solution

Acetic acid, glacial --------------------------------------------------------------                3.0 ml

Distilled water -------------------------------------------------------------------        97.0 ml

1% Alcian Blue Solution

Alcian blue 8 GX, C.I. 74240 ---------------------------------------------------                1.0 gm

3% acetic acid -------------------------------------------------------------------        100.0 ml

       Filter and add a few crystals of thymol.

0.5% Periodic Acid Solution

Periodic acid --------------------------------------------------------------------                0.5 gm

Distilled water -------------------------------------------------------------------        100.0 ml

Modified Lillie’s Schiff Solution

(see PAS method)

0.3% Sodium Borate

Sodium borate (Na2 B4 O7 10H2O) ---------------------------------------------                0.3 gm

Distilled water -----------------------------------------------------------------                100.0 ml

1. Deparaffinize and hydrate to distilled water.  For plastic sections begin at Step 2.
2. Place in 3% acetic acid for three minutes.

3.        Place in 40 ml of 1.0% alcian blue solution in a glass Coplin jar and microwave at power level 1 (100W) for 3 minutes.  Dip the slides up and down several times and allow them to remain in the hot solution for 5 minutes.        

4.        Rinse in three changes of distilled water.

5. Place in 0.5% periodic acid for 15 minutes.
6. Rinse with four changes of distilled water.
7. Place in modified Lillie’s Schiff Solution for 30 minutes.
8. Rinse in four changes of distilled water.
9. Place in 0.3% sodium borate for 15 seconds.
10. Rinse in four changes of distilled water.
11. Dehydrate in graded alcohols.
12. Clear in three or four changes of xylene.
13. Mount with synthetic resin.

Normal and abnormal tubular and glomerular basement membranes, Bowman’s capsule, elastic lamina of blood vessels, some renal casts, and juxtaglomerular granules are red.  Epithelial cell mucoproteins and acid mucopolysaccharides in blood vessels are blue.

When heating the alcian blue solution with microwave irradiation the top portion of the solution is warmer by 10-15o C than that near the bottom of the Coplin jar.  Therefore, in order to equalize the temperature of the solution the slides are dipped up and down.  This assures uniformity of staining throughout the tissue sections.

The stain may be performed at room temperature by staining the sections in the alcian blue solution for 30 minutes.

Garvey, W.:  Combined modified periodic acid-Schiff and batch staining method.  J. Histotechnol. 15:117-120, 1992.

Lillie, R.D. and Fullmer, H.M.:  Histopathologic Technic and Practical Histochemistry, 4th ed., New York, McGraw-Hill, 1976, pp. 307-308.

McManus, J.F. and Mowry, R.W.:  Staining Methods Histologic and Histochemical, New York, Paul B. Hoeber, 1960, pp. 63-64.