For counting and subsequent analysis, the macrophages were separated into total monocytes and macrophages (labeled by combined ED1 and ED2 immunohistochemistry) and total resident macrophages (all ED2+ cells). In the previous study, the ED2+ resident macrophages represented 80% of the total testicular mononuclear phagocyte population. Intratesticular ED1+ED2- monocyte/macrophages and putative ‘‘long-term resident’’ macrophages (ED2+ED1- cells) were calculated by subtraction, as previously described. birth control pills
Serum samples were assayed for LH and FSH by specific RIAs, as previously described. Assay sensitivity was 0.09 ng/ml for LH and 1.5 ng/ml for FSH. Testicular IF and homogenates were assayed for testosterone without extraction using a direct 125I-testosterone RIA, or after extraction with chloroform-hexane using a [3H]testosterone RIA. Extraction recoveries were 78-82%.
Testicular IF was assayed for immunoreactive MIF by a sandwich ELISA employing a monoclonal anti-MIF capture antibody, a rabbit polyclonal anti-MIF detector antibody, and a purified recombinant mouse MIF standard, as previously described. Note that some samples were not available for MIF assay because of limitations on sample volumes.