Uplsapo 20xo

Samples were incubated overnight at 4 °C with a monoclonal anti-RBPMS antibody (1:500, rabbit; ABN1362, Merck Millipore) for the retina^{31 (link)}, with a monoclonal anti-NeuN antibody (1:500, mouse, clone A60; MAB377, Merck Millipore) for brain sections^{48 (link)}. The sections were then incubated with secondary antibodies conjugated with Alexa Fluor 488 (1:500, donkey anti-mouse and donkey anti-rabbit IgG 488, polyclonal; A-21202 and A-21206, Invitrogen, respectively) and DAPI (1:1,000; D9542, Merck Millipore) for 1 h at room temperature. An Olympus FV1000 confocal microscope with ×20 objective (UPLSAPO 20XO with a numerical aperture of 0.85) was used to acquire the images of flat-mounted retinas and brain sections (FV10-ASW v. 4.2 software).
On the confocal images processed with Fiji (ImageJ v. 1.53q), RBPMS- and NeuN-positive cells were automatically counted with the ‘analyze particles’ plugin. The cells were manually counted by two different users, with the ‘cell counter’ plugin. Quantification was performed by acquiring confocal stacks in at least four randomly chosen transfected regions of 0.4 mm² (Extended Data Fig. 1). For V1 neurons, the sagittal brain slice with the largest tdTomato fluorescence zone was selected for each animal. A region of interest was manually defined in V1 and the quantifications were performed in at least six randomly chosen regions of 0.4 mm².

Following electrophysiological recordings, rats were euthanized, and their brains were extracted and fixed by overnight incubation in 4% paraformaldehyde (100496, Sigma-Aldrich) at 4 °C. Brains were then cryoprotected in 30% sucrose (84097, Sigma-Aldrich) and 50 µm sagittal slices were cut with a microtome (HM450, Microm). The slices with the most intense tdT fluorescence from each brain were selected for further immunohistochemistry and imaging. Cryosections were permeabilized by incubation with 0.5% Triton X-100 in PBS for 1 h at room temperature and were then incubated in blocking buffer (PBS + 1% BSA + 0.1% Tween 20) for 1 h at room temperature. Samples were incubated overnight at 4 °C with monoclonal anti-NeuN antibody (1:500; Mouse, MAB377, Merck Millipore), in a 50% dilution of blocking buffer + 0.5% Triton X-100. Secondary antibodies conjugated with Alexa Fluor dyes (1:500; Molecular Probes) and DAPI (1:1000, D9542, Merck Millipore), were incubated with the samples for 1 h at room temperature. An Olympus FV1000 laser-scanning confocal microscope with a 20× or 40× objective (UPLSAPO 20XO, NA: 0.85) was used to acquire images of brain sections.