Multiple epitope labeling by the exclusive use of alkaline phosphatase conjugates in immunohistochemistry

Here we demonstrate a simple and reliable multiple epitope labeling technique based exclusively on the alkaline phosphatase (AP) enzyme-linked visualization method. AP is functionally blocked by ethylenediaminetetraacetic acid (EDTA), which allows the repeated use of AP conjugates in immunohistochemistry with different substrates. We found that reactivation of AP function following EDTA incubation is dependent on EDTA concentration and incubation time. While incubation times of up to 2 h in 0.25 M EDTA, pH 6, exhibit a resumption of AP enzyme function? more than 2 h of incubation irreversibly blocks AP enzyme activity. Surprisingly, EDTA incubation also results in considerable but not complete inhibition of antibody crossreactivity during immunohistochemistry. Thus, this technique is suitable for single-layer, multiple-staining experiments with AP-linked primary antibodies or multilayer labeling with antibodies of various species for sequential staining rounds. We demonstrate the applicability of this technique in immunohistochemistry by double-labeling experiments using the monoclonal antibodies anti-glial fibrillary acidic protein, anti-leucocyte common antigen, anti-CD43/CD45RA (pan-human leucocyte), and anti-migration inhibitory factor-related protein-8 in combination with an in situ nick translation assay to characterize differentiating antigens of apoptotic cells in human glioblastoma paraffin sections.