Alexa fluor 647 conjugated isolectin gs b4

Anesthetized mice were perfused with 20 mL of PBS. Eyes were enucleated and fixed in 4% paraformaldehyde in 2× PBS for 15 min and then transferred to 2× PBS on ice for 10 min. After dissecting eyes, retinal whole mounts were prepared. Retinas were then transferred to ice-cold methanol and kept at −80 °C until use.
For immunohistochemistry, retinas were first blocked in a blocking buffer (0.3% Triton, 0.2% BSA, and 5% goat serum in PBS) for 1 h at room temperature and incubated with primary antibodies and Alexa Fluor 647-conjugated isolectin GS-B4 (1:100; Thermo Fisher Scientific) overnight at 4 °C. After washing, retinas were incubated with secondary antibodies for 4 h at 4 °C. Retinas were mounted after washing. Rabbit anti-P2ry12 antibody (1:500; a gift from H. Weiner, Brigham and Women’s Hospital), rat anti-CD11b antibody (1:100, clone M1/70; Abcam), rat anti-MHC class II (1:1,000, I-A/I-E; BD Pharmingen), rat anti-CD4 (1:200, RM4-5; BD Pharmingen), and rat anti-CD8a (1:200, 53-6.7; BD Pharmingen) were used for primary antibodies. Alexa Fluor 594-conjugated goat anti-rabbit antibody, and Alexa Fluor 488-conjugated goat anti-rat antibody (1:500; Thermo Fisher Scientific) were used for secondary antibodies.

After anesthesia, eyes were enucleated following retinal perfusion with PBS. The eyes were fixed in 4% paraformaldehyde (PFA) in 2× PBS for 15 min and then were transferred to 2× PBS on ice for 10 min. After the eyes were dissected, retinal whole mounts were prepared. The detached area was easily distinguishable by the characteristic morphology. The retinas were then transferred to ice-cold methanol and kept at −80 °C until use. For IHC, rabbit anti-P2ry12 Ab (1:500; a gift from H.L.W.), rat anti-CD11b Ab (1:100, clone M1/70; Abcam), and rat anti-F4/80 Ab (1:2,000, clone CI:A3-1; Bio-Rad) were used for primary antibodies, and Alexa Fluor 594-conjugated goat anti-rabbit Ab and Alexa Fluor 488-conjugated goat anti-rat Ab (1:500; Thermo Fisher Scientific) were used for secondary antibodies. The retinas were first blocked in a blocking buffer (0.3% Triton X-100, 0.2% BSA, and 5% goat serum in PBS) for 1 h at room temperature and were incubated with primary antibodies and Alexa Fluor 647-conjugated Isolectin GS-B4 (1:100; Thermo Fisher Scientific) overnight at 4 °C. After washing, the retinas were incubated with secondary antibodies for 4 h at 4 °C. The retinas were mounted after washing.