After this blocking procedure, the sections were rinsed again in PBS-1% BSA and then incubated either with the rabbit anti-rat MCP-1 antibody or with a monoclonal antibody against either rat monocytes/macrophages or differentiated macrophages (1:200 dilution, 30 min at 37°C). After the incubation with primary antibody, the sections were rinsed in PBS-0.1% BSA and then exposed to either biotinylated goat anti-rabbit (MCP-1) or goat anti-mouse (monocytes/macrophages, differentiated macrophages) immunoglobulin (1:200 dilution; 30 min at 37°C). Detection of the antigen-antibody complex was achieved by using a Vector avidin-biotin-peroxidase kit and 3-amino-9 ethyl-carbazole as the substrate.
The tissue sections were counterstained with hematoxylin, rinsed in distilled water, and dipped in Scott’s tap water (a mordant) before being mounted with aqueous mounting medium. MCP-1 staining was graded for intensity and for distribution by two independent observers using coded slides. Buy Advair Diskus Online
Nonspecific staining was assessed by replacement of primary or secondary antibodies with the appropriate serum and was undetectable in all instances.