Fitc conjugated goat anti rat secondary antibody

Eyeballs were removed (n = 3 per group and time) at 3 days PI from C57BL/6 mice, immersed in 0.01-M PBS, embedded in OCT compound (Tissue-Tek; Sakura Finetek, Torrance, CA, USA), and frozen in liquid nitrogen. Ten-micrometer sections were cut, mounted to poly-l-lysine-coated glass slides, and stored at 37°C overnight. After a 5-minute fixation in acetone, slides were blocked with 0.01-M PBS containing 10% blocking serum for 30 minutes at room temperature. The sections were incubated at a 1:100 dilution of the rat anti-F4/80 antibodies (Abcam) at 4°C overnight. This was followed by FITC-conjugated goat anti-rat secondary antibody (1:500, 1 hour at room temperature without light; Abcam). Isotype IgG was used as the negative control. Finally, sections were visualized and digital images captured with a Zeiss Axio Vert microscope (Carl Zeiss Microscopy, Jena, Germany) at 40× magnification.