Immunofluorescence of Retinal Sections

Immunofluorescence of retinal sections was performed as previously reported [33 (link), 37 (link)]. The eyecups were cut into 10 μm thick sections. The cryosections were, respectively, incubated with monoclonal mouse anti-mouse A_2AR antibody (05-717; Millipore, USA), polyclonal rabbit anti-mouse Iba-1 antibody (019-19741; Wako, Japan), monoclonal rat anti-mouse CD11b antibody (ab8878; Abcam, USA), monoclonal rat anti-mouse MHC Class II antibody (ab25333; Abcam, USA), monoclonal mouse anti-mouse GFAP antibody (mab3402; Sigma-Aldrich, USA), polyclonal rabbit anti-mouse GFAP antibody (ab7260; Abcam, USA), and polyclonal rabbit anti-mouse IL-1β antibody (ab9722; Abcam, USA) by overnight incubation at 4°C. The cryosections were then, respectively, incubated in Alexa 555-conjugated anti-mouse IgG (4409), Alexa 488-conjugated anti-mouse IgG (4408), Alexa 555-conjugated anti-rabbit IgG (4413), Alexa 488-conjugated anti-rabbit IgG (4412), or Alexa 488-conjugated anti-rat IgG (4416) (all from cell signaling technology, Inc.). The sections were finally counterstained with DAPI (sc-3598; Santa Cruz Biotechnology, Inc.). Images were captured with a confocal microscope (SP5; Leica Microsystems, Inc., USA) with a fixed detection gain for each comparative section.