G9269

Cryosections were blocked in 10% horse serum, 0.1% Triton X-100 in PBS (PBT), pH 7.5, for 1 h before adding primary antibodies diluted in blocking solution. The following primary antibodies were used (anti-): pAkt^Ser473 (1:500; Cell Signaling #4060), pS6^Ser235/236 (1:500; Cell Signaling #4856), Brn3b (1:500; Chemicon #AB5945), Chx10 (1:200; Santa Cruz #sc-21690), cone-arrestin (1:500; Millipore #AB15282), Pax6 (1:500; Covance Research #PRB-278P), Pten (1:500; Cell Signaling #9559), rhodopsin (1:500; Chemicon #MAB5356), calbindin (1:500; Sigma #C9848), GFAP (1:500; Sigma #G9269), CRALBP (1:500; Abcam #ab15051), glutamine synthetase (1:500; Abcam #ab73593), fibronectin (1:200; Abcam #ab2413), laminin (1:500; Sigma #L9393), RPE65 (1:500; ORIGENE #TA309839), Otx2 (1:500; Abcam #ab21990), N-cadherin (1:200; BD Transduction Labs #610920), ZO-1 (1:100; ThermoFisher Scientific #33-9100) and Sox9 (1:500; Millipore #AB5535). Slides were incubated in primary antibodies overnight at 4°C. The next day, slides were washed three times in PBT before incubating with secondary antibodies conjugated with Alexa Fluor 568 (1:500; Molecular Probes) or Alexa Fluor 488 (1:500; Molecular Probes) for 1 h. Slides were then washed three times in PBT before labelling nuclei with DAPI.

Primary antibodies used were as follows: rabbit anti-GFAP (1:200; Sigma-Aldrich, catalog #G9269; RRID: AB_477035) and biotinylated goat anti-GFP (1:400; Abcam, catalog #ab6658; RRID: AB_305631). Secondary antibodies used were as follows: streptavidin Alexa 488 (1:800; Invitrogen, catalog #S32354; RRID: AB_2315383) for GFP. Goat anti-rabbit antibody conjugated to Alexa 568 (1:400; Thermo Fisher Scientific, catalog #A11011; RRID: AB_143157) for GFAP. Tissue processing for immunohistochemistry was performed as described by Subramanian et al. (2011) (link). Immunohistochemistry for calculating percentage astrocytes in electroporated brains was performed in three biological replicates.