Retinal Flatmount Staining for Astrocytes and Microglia

GFAP and isolectin B4 double staining of the retinal flatmounts were conducted as previously described [56 (link),57 (link),58 (link)]. Briefly, retinal flatmounts were washed in ice-cold PBS/Triton X-100 (TXPBS), fixed in methanol, followed by permeabilization and blocking in PermBlock (PBS + 0.3% Triton X-100 + 0.2% bovine serum albumin) in 5% goat serum for 1 hour. After washing in TXPBS, flatmounts were incubated with rabbit GFAP primary antibody (Cell Signaling Technologies, Danvers, MA, USA) overnight at 4°C. Following several washes with TXPBS, the flatmounts were incubated with Alexa Fluor 488 goat anti-rabbit fluorescent secondary antibodies, and Alexa Fluor 594 Isolectin B4 (ThermoFisher Sci/Life Technologies, Grand Island, NY, USA) overnight at 4 °C. The flatmounts were washed with TXPBS and mounted on slides with prolong anti-fade fluorescent mounting media and imaged at 20X magnification using the Olympus BX53 microscope, DP72 digital camera, and CellSens Dimension imaging software (version 2.1) from Olympus America, Inc. (Center Valley, PA, USA), attached to an HP Z44 computer.