THE EFFECT OF FIXATION AND TRYPSINIZATION ON THE IMMUNOHISTOCHEMICAL DEMONSTRATION OF INTRACELLULAR IMMUNOGLOBULIN IN PARAFFIN EMBEDDED MATERIAL

Abstract

Using an indirect labeled immunoperoxidase technique the influence of fixation time on the antigenicity of intracellular Ig in [human] lymphoid tissue fixed in buffered formalin was investigated. Within a fixation period of 96 h a decrease of 15% of stainable Ig containing cells was found, for every 24 h the fixation time was prolonged. By comparing sections from tissue fixed in buffered formalin and selected fixatives (Lillie''s AAF, Bouin''s fluid, Clarke''s fluid and 96% ethanol 1% acetic acid (E-A) processed at 4.degree. C and at 25.degree. C an increased number of stained Ig containing cells was found in tissue fixed in Lillie''s AAF, Bouin''s fluid, Clarke''s fluid and E-A processed at 4.degree. C. No difference was found between tissue fixed in buffered formalin and E-A processed at 25.degree. C. In addition the effect of pretreatment of the sections with trypsin on the number of stainable Ig containing cells was investigated. Trypsinization of sections from formalin fixed material increased the number of stainable cells substantially. No essential effect was seen on tissue fixed in Lillie''s AAF and Bouin''s fluid. In contrast trypsin treatment of sections from tissue fixed in Clarke''s fluid and E-A completely destroyed the tissue. No differences were observed between different Ig classes examined in the effect of fixation time, fixatives and trypsinization.