Rabbit anti gfap polyclonal antibody

After sacrifice, the eyeballs were prepared in paraffin and processed for retinal sections described above. Next, the sectioned retinal pieces were immunohistochemically processed utilizing the following primary antibodies: goat anti-ChAT polyclonal antibody (Millipore, CA, USA), rabbit anti-GFAP polyclonal antibody (Millipore, CA, USA) and mouse anti-vimentin monoclonal antibody (Sigma-Aldrich, MO, USA). Thereafter, the retinal sections were soaked in the appropriate secondary antibodies: rhodamine-bound rabbit anti-goat antibody (Millipore, CA, USA), fluorescein isothiocyanate (FITC)-bound goat anti-rabbit IgG (Millipore, CA, USA) or FITC-bound goat anti-mouse IgG (Millipore, CA, USA). Simultaneously, the cellular nucleus was labeled with 4,6-diamidine-2-phenylindole dihydrochloride (DAPI; EMD Chemicals, Darmstadt, Germany) as described previously [1 (link), 24 (link)]. A fluorescence microscope was used to examine the sectioned retinal pieces as described previously [1 (link), 24 (link)]. To grade the immunoreactivity levels in the retinal sections among the various experimental groups, a researcher who was blinded to the status of the sections was requested to compare the immunoreactivity level of the various experimental groups against the sham group (control).

After sacrifice of the animals and intracardial perfusion, the eyeballs of the rats were enucleated, fixed for 45 min, dehydrated and finally embedded in paraffin as above-mentioned. Sampling was carried out 1 day after the sham procedure or induction of retinal ischemia with pre-ischemia/post-ischemia administration of DNL or vehicle. Each 5 μm retinal section was incubated overnight with primary antibodies, namely either goat anti-ChAT polyclonal antibody (1:100; AB144p; Chemicon), mouse anti-vimentin monoclonal antibody (1:100; V6630; Sigma-Aldrich), or rabbit anti-GFAP polyclonal antibody (Millipore). Next, retinal sections were incubated with an appropriate secondary antibody, either rhodamine-conjugated rabbit anti-goat antibody (1:500; AP106R; Chemicon), or fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (1:500; AP124F; Millipore)/anti-rabbit IgG (AP132F; 1:500; Millipore). In parallel, cellular nuclei were labeled using 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes). Finally, retinal sections were examined using a fluorescence microscope (Olympus BX61) by an expert who was masked to the conditions under which the sectioned samples had been obtained in order to grade the immunolabeling level of various different groups against the control group (sham).