Factors affecting the sensitivity and specificity of the cytochemical reaction for the anti-horseradish peroxidase antibody in lymph tissue sections.

Factors which increase the sensitivity and specificity of the cytochemical reaction for the antibody to horseradish peroxidase (HRP) in precursors of plasma cells and in lymphocytes were studied in sections of popliteal lymph nodes of rats. The lymph nodes were removed 3-5 days after a secondary injection of HRP into the footpads and were fixed for 5 hr in a 4% cold formaldehyde solution (Straus W: Histochemistry 53:273, 1977). Brief postfixation of the frozen sections with cold acetone improved the retention of the antigen at the sites of the antibody in the precursor cells, and it improved the quality of fixation without appreciably weakening the antigen-binding capacity of the antibody. The cytochemical reaction for the anti-HRP antibody was intensified by staining with diaminobenzidine (DAB) and H2O2 at pH 5-6, or by staining at pH 7.4 in the presence of imidazole. Imidazole partially inhibited endogenous peroxidase activity. Pretreatment with phenylhydrazine prevented nonspecific background adsorption of HRP. Phenylhydrazine had the additional advantage of inhibiting most of the endogenous peroxidase activity (Straus W: J Histochem Cytochem 20:949, 1972). The intensity of the antibody reaction in the proplasma cells developing in the medullary cords varied greatly depending on the stage of maturation from lymphocytes and blast cells. Many lymphocytes in the cortex of the lymph node showed a strong perinuclear antibody reaction when the tissue sections were postfixed with cold acetone, and the peroxidase complexed to the antibody was visualized by staining with DAB and H2O2 at pH 5-6. The antibody reaction also occurred at the surgace of many lymphocytes when the tissue sections, postfixed with cold acetone, were stained with DAB and H2O2 at pH 7.4 in the presence of imidazole. Other lymphocytes showed a strong surface, perinuclear, and cytoplasmic antibody reaction after staining at pH 5-6 as well as after staining at pH 7.4, while yet other lymphocytes remained unstained.