Enzyme-labeled Antigen Method: Histochemical Detection of Antigen-specific Antibody-producing Cells in Tissue Sections of Rats Immunized With Horseradish Peroxidase, Ovalbumin, or Keyhole Limpet Hemocyanin

The enzyme-labeled antigen method is a histochemical technique that visualizes antigen-specific antibody-producing cells in tissue sections, originally documented in 1968. In this study, we attempted to reemerge this hidden but potentially useful method in rat models immunized with horseradish peroxidase (HRP), ovalbumin (OA), or keyhole limpet hemocyanin (KLH). After repeated immunization in footpads, popliteal, groin, and axillary lymph nodes and spleen were sampled. Paraformaldehyde-prefixed frozen sections were incubated with HRP, biotinylated OA, or biotinylated KLH. Proteinase K pretreatment and the secondary use of HPR-labeled streptavidin were applied in the latter two situations. Plasma cells producing antigen-specific antibodies were visualized. Proportions of antigen-specific antibody-producing cells in total plasma cells shown with the immunoperoxidase method for rat immunoglobulins were evaluated. The percentage of antigen-specific plasma cells reached ∼50% of total plasma cells in the regional lymph nodes. The specificity was confirmed by (a) negativity in non-immune rat tissue, (b) negativity with indifferent antigen probes, and (c) abolishment of the reactivity with the corresponding rat serum. In buffered formalin-fixed, paraffin-embedded tissues, fewer plasma cells were labeled for HRP and KLH antibody reactivity after strong proteolysis and prolonged incubation. Expectedly, this method allows us to observe antigen-specific antibody-producing cells under varied pathological conditions.