Microwave Periodic Acid-Schiff with Diastase (DiPAS)

MICROWAVE PERIODIC ACID-SCHIFF WITH DIASTASE

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Mucin in intestine (upper left), hepatocytes in liver with amyloidosis (upper right), intracytoplasmic hyaline globues in liver of alpha-1-antitrypsin deficiency (lower left), and coccidioides in lung (lower right). Small intestine (Courtesy of Dr. Zhenhong Qu).

2ndPAS-D

Diagnostic Application:

Tumor of glandular origin (e.g., adenocarcinoma) contains polysacride glycoprotein stained pink or red.

Highlights alpha-1-antitrypsin in hepatocytes, a feature of alpha-1-antitrypsin deficiency.

Many fungal microorganisms are also stained by this methods.

Material and Solutions:

FIXATION: Absolute alcohol or 10% formalin - alcohol is preferred. If 10% buffered neutral formalin is used, there will be a significant loss of glycogen.

TECHNIQUES: Paraffin sections cut at 4 μm.

SOLUTIONS:

Phosphate Buffer Solution pH 6.0*

Sodium chloride ----------------------------------------------------------------- 8.000 gm

Sodium phosphate, dibasic Na2HPO4 -------------------------------------------- 0.282 gm

Sodium phosphate, monobasic NaH2PO4 · H2O --------------------------------- 1.970 gm

Distilled water ------------------------------------------------------------------- 1000.000 ml

*The pH of the solution without the sodium chloride is 6.0.

With the sodium chloride the pH will be approximately 5.7.

Diastase Digestion Solution

Phosphate buffer solution ------------------------------------------------------ 40.00 ml.

Diastase of malt ----------------------------------------------------------------- 0.04 gm

0.5% Periodic Acid Solution

(See PAS Method)

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

Staining Procedure:

Run two slides. Diastase digest one of the slides and then do the PAS stain on both slides.

1. Deparaffinize and hydrate to distilled water.

2. Place in diastase digestion solution in a glass Coplin jar. Place in a microwave oven and microwave at power level 1 for one minute. Transfer to a 37o C oven for 30-60 minutes. Leave the PAS slide in distilled water.

3. Wash in tap water for 3 minutes and rinse in distilled water, two changes.

4. Place in 0.5% periodic acid for 10 minutes.

Rinse in four changes of distilled water.
Place in modified Lillies Schiff solution for 15 minutes.

7. Rinse in four changes of distilled water.

8. Place in 0.3% sodium borate for 15 seconds.

9. Rinse in four changes of distilled water.

10. Stain in acidified Lillie-Mayer hematoxylin for 45 seconds.

11. Rinse in three changes of distilled water.

12. Blue hematoxylin in 0.3% sodium borate for 15 seconds.

13. Rinse in four changes of distilled water.

14. Dehydrate in graded alcohols.

15. Clear in three or four changes of xylene and mount with synthetic resin.

Staining Results:

Staining attributable to glycogen is selectively eliminated in the diastase digested slide, but will be stained magenta in the undigested slide.

Nuclei --------------------------------------------------------------- blue

References:

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

Lillie, R.D.: Histopathologic Technic and Practical Histochemistry, 3rd Edition, New York, McGraw-Hill, 1965, pp. 496-497.

Luna, L.G.: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd Edition, New York, McGraw-Hill, 1968, pp. 159, 171.