Immunohistochemistry and Stereological Analysis
Sections were stained with the mouse anti-rat monoclonal antibodies ED1, which labels a lysosomal antigen specific to monocytes, dendritic cells, and some macrophages (1:4 dilution), or ED2, which labels resident macrophages specifically (1:800 dilution), using immunoperoxi-dase detection and subsequent hematoxylin counterstaining as previously described. In order to label all cells of the mononuclear phagocyte and dendritic cell lineages, sections were double-stained with ED1 and ED2 combined. Since dendritic cells represent only a very small cell population compared with the number of mononuclear phagocytes in most tissues, and this also appears to be true of the testis, this ED1+ and ED2+ double staining provides an estimate of the total testicular monocyte/macrophage population. ventolin inhalers
Immunostained cell profiles containing a nuclear profile were counted using an Olympus (Tokyo, Japan) BH-2 microscope linked to an Amiga (Bensheim, Germany) 2000 computer with an Impact Vision-24 (G.V.P., King of Prussia, PA) professional video adaptor, as previously described. Briefly, the GRID (Interactivision, Silkeborg, Denmark) software package was used to generate an unbiased counting frame that was superimposed on the microscope image. Fields were sampled with a systematic uniform random scheme using a motorized stage. The numerical density of nuclei associated with immunopositive cytoplasm was calculated and was used to determine the total numbers of cells per testis.