Immunofluorescence and Histological Analysis of Rat Retina

Anesthetized rats were perfused transcardially with pre-cooled phosphate-buffered saline. Intact eyes were enucleated and fixed in FAS eyeball fixative solution (G1109; Servicebio, Beijing, China) for 24 h at room temperature. After fixation, eyeballs were dehydrated in ethanol and embedded in paraffin. Sections (3 μm) were made around the optic nerve for further immunofluorescence staining and hematoxylin and eosin (HE) staining. Dewaxed and antigen-retrieved paraffin retinal slices were applied to immunofluorescence, as described in our previous study (20 (link)). Three antibodies (all from Abcam, Cambridge, UK) were used: anti-PPARα (ab215270; 1:100), anti-glial fibrillary acidic protein (GFAP; ab33922; 1:300), and anti-cyclooxygenase 2 (COX2; ab62331; 1:100). Retina HE staining was performed by the Hematoxylin-Eosin/HE Staining Kit (G1120; Servicebio, Wuhan, China) according to the manufacturer's instructions.