Restoration of antigenicity of tissue antigens, cell-bound immunoglobulins and immune deposits in paraffin-embedded tissue. The influence of fixation and proteolytic enzymatic digestion.

The possibility of preservation and restoration of antigenicity of some antigens in paraffin-embedded tissue was evaluated by direct immunofluorescent technique on deparaffinized sections. Fixation with 96% ethanol-1% acetic acid, 10% neutral buffered formalin and p-formaldehyde was useful for the preservation of tissue antigens and immune deposits, whose antigenicity could be easily restored by trypsin digestion. Neutral buffered formalin was also a satisfactory fixative in immunofluorescent staining on lymphocyte/plasma cell-bound immunoglobulins. Fixation with alcohol-Bouin's fluid showed contrast results; feasible for staining of cell-bound immunoglobulins, but poor for that of glomerular immune deposits. After papain digestion, BSA and lysozyme, antigens of immune complexes, were easily detected in experimental chronic serum sickness glomerulonephritis. Pepsin was more efficient than trypsin in restoring the antigenicity of renal tissue antigens such as fibronectin and polyantigenic basement membrane, but the brush border antigen of the proximal renal tubules was frail to the pepsin digestion. In general, the enzymatic digestion time necessary for the restoration of antigenicity was in parallel with fixation time. Results obtained have shown that deparaffinized sections could be used as satisfactory substrate for immunohistochemistry when proper fixation and efficient proteolytic enzymatic pretreatments were performed.