Representative animals were selected for analysis. All the rats used in the experiment were initially inoculated subcutaneously with an amount of 50,000 NS1 rat glioma cells. Tumors isolated from the rats were fixed using Phosphate-buffered 4% Paraformaldehyde as described previously [3 (link)], paraffin embedded and sectioned using a microtome. The primary polyclonal antibodies were diluted 1:400 in PBS containing 1% BSA and 2% normal goat serum in the following manner; the fixed tissue samples were incubated with rabbit anti-rat C1 inactivator (Covance, USA), permeabilized with 0.1% Triton™ X-100 for 10 minutes, blocked with 1% BSA for 1 hour and labeled with 2 μg/mL the rat primary antibody for 1 hours at room temperature. The tissue was subsequently washed and incubated for 1 hour at room temperature with secondary antibodies consisting of Alexa Fluor 594-conjugated goat anti-rabbit serum (ab150084 Abcam) at a concentration of 0.5μg/mL in phosphate buffered saline containing 0.2% BSA at room temperature. After washing with PBS, the tissue was mounted with anti-fading vecta-shield mounting medium with 4,6-diamidino-2-phenylindole (nuclear stain with DAPI) (Vector Laboratories Inc., Burlingame, USA) and were photographed using fluorescence microscope fitted with the appropriate wavelength filters.
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