Immunofluorescence Analysis of Retinal Microglia

We verified the purity of primary murine retinal microglia cultures and the localization of GPER in the microglia using immunofluorescence. Briefly, the retinal microglial cells were fixed with 4% paraformaldehyde, and then permeabilized with 0.5% Triton X-100 at room temperature for 15 min. Then, the cells were incubated with 6% normal goat serum (Hyclone, Logan, Utah, USA) at room temperature for 30 min for blocking. The slides were then incubated overnight with either mouse anti-CD11c antibody (1:200, Abcam, Cambridge, UK), a microglia specific marker located on their cell membrane [50 (link)], rabbit anti-GPER antibody (1:200, Abcam, Cambridge, UK), or PBS (negative control) at 4°C. The cells were then incubated with the Alexa Fluor 488-tagged secondary antibody (1:800, Abcam, Cambridge, UK) for 30 min at 37°C in the dark. Then, after staining the nuclei with DAPI (Beyotime, Shanghai, China), the slides were washed thrice with PBS to remove the excess DAPI. The slides were sealed with anti-fluorescence quencher (Beyotime, Shanghai, China) and imaged using a laser confocal fluorescence microscope (Leica, Solms, Germany). The average fluorescent intensity was measured using the Image J software (version 1.46, National Institutes of Health, Bethesda, MA, USA).