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PRINCIPLE OF THE TEST.
Used in immmunohistochemistry for proteolytic digestion.
SPECIMEN REQUIRED.
All human tissue excised at surgery, outpatient clinics, and postmortems,
fresh or in fixative, along with a filled-out Tissue Examination Form
(U. S. Standard Form 515, USSF515).
REAGENTS, INSTRUMENTATION.
0.4% pepsin solution.
Pepsin.
0.2N HCl.
95% ethanol.
3% hydrogen peroxide.
Absolute methanol.
STEP-BY-STEP DESCRIPTION.
SOLUTIONS.
A. Pepsin 40mg
B. 0.2N HCl 10ml
1. Deparaffinize sections and bring to water.
2. Treat sections with 0.4% pepsin in 0.2N HCL
in a 37oC oven 20 minutes.
3. Rinse sections thoroughly (5 minutes) with running tap water.
4. Dehydrate in 95% ethanol.
5. Block endogenous peroxidase staining with 3% hydrogen peroxide
in absolute methanol (15 minutes).
6. Rinse in running tap water 3 - 5 minutes, then PBS buffer
for 5 minutes.
7. Start immunostains.
REFERENCES.
1. Miller KD.
Immunohistochemical Techniques. Chapter 21. pp. 421-464.
In: Bancroft JD, Gamble M.
Theory and Practice of Histological Techniques. Fifth Edition.
Edinburgh: Churchill Livingstone. 2002;:421-464.
ISBN 0-443-06435-0, 796 pages.
2. Prophet EB, Mills B, Arrington JB, Sobin LH, eds.
Laboratory Methods in Histotechnology.
Washington, DC: Armed Forces Institute of Pathology. 1992;:.
ISBN 1-881041-00-X, 278 pages.
3. Mikel UV, ed.
Advanced Laboratory Methods in Histology and Pathology.
Washington, DC: Armed Forces Institute of Pathology. 1994;:.
ISBN 1-881041-13-1, 254 pages.