Goat anti brn3a

To analyze structural changes to the retina, eyes from animals with three weeks IOP elevation post Ad5.MYOC injection and from control mice were fixed in 4% paraformaldehyde for two hours and rinsed in PBS. After cryopreservation using sucrose embedding, sagittal 7 µm sections were washed three times for 5 min in PBS and blocked in 1%BSA/PBS for 1 h at room temperature. Sections were incubated with rat-anti C1q (Abcam, Cambridge, UK, diluted 1:50 in 1%BSA/PBS), goat-anti Brn3a (Santa Cruz, Dallas, TX, USA, diluted 1:200 in 1%BSA/PBS), goat-anti PSD95 (Abcam, diluted 1:1000 in 1%BSA/PBS), or rabbit-anti GABA_A Receptor A1 (Millipore Sigma, Burlington, MA, USA, 1:500 in 1%BSA/PBS) at 4 °C overnight. Then, slides were washed and incubated for three hours at room temperature in goat anti-rat Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA, 1:500 in PBS) donkey anti-goat Alexa Fluor 546 (Invitrogen, 1:500 in PBS) or donkey anti-rabbit Alexa Fluor 488 (Invitrogen, 1:500 in PBS). Slides were washed three times for 5 min in PBS followed by a rinse in 70% EtOH and distilled water. After coverslipping using Aquamount, dry slides were imaged on a Zeiss 710 confocal microscope (Carl Zeiss, Thornwood, NY, USA).

Rats were euthanized at one or two weeks post-axotomy by transcardial perfusion with 4% PFA and both the left (optic nerve lesion) and right (intact control) retinas were dissected and fixed for an additional 15 min. Brn3a immunodetection on whole-mounted retinas was performed as described [27] (link). Briefly, whole mounted retinas were permeabilized in PBS containing 0.5% Triton X-100 (Fisher, Waltham, MA) by freezing them at −80°C for 15 min, rinsed and incubated overnight at 4°C with goat-anti-Brn3a (0.27 μg/ml, Santa Cruz Biotechnologies, C-20) in blocking buffer (PBS, 2% normal donkey serum, 2% Triton X-100). Retinas were washed and incubated for 2 hrs at room temperature with Alexa Fluor donkey anti-goat IgG (1 μg/ml, Jackson ImmunoResearch Laboratories Inc.). Retinas were then rinsed, mounted vitreal side up, and covered with anti-fade solution (SlowFade, Molecular Probes, Eugene, OR). Brn3a-labeled neurons were counted within three square areas at distances of 1, 2 and 3 mm from the rat optic disc in each of the four retinal quadrants for a total of twelve retinal areas. Fluorescent staining was examined with a Zeiss Axioskop 2 Plus microscope (Carl Zeiss Canada, Kirkland, QC). Images were captured with a CCD video camera (Retiga, Qimaging, Burnaby, BC) and analyzed with Northern Eclipse software (Empix Imaging, Mississauga, ON).

Following the treatments, animals were euthanized and their eyes were enucleated. The eye cups were fixed overnight at 4°C in 4% paraformaldehyde (PFA) and retinal flat mounts were prepared. To prevent non-specific binding with the secondary antibody, blocking was carried out overnight at 4°C using 5% normal donkey serum and 5% BSA in PBS. Retinal flat mounts were incubated in primary antibody solution, goat anti-Brn3a (1:200; Santa Cruz) for 72 hours at 4°C. Subsequently, secondary antibody incubation was carried out using a 1:1000 dilution of Alexa 647 conjugated donkey anti-goat antibody (Life Technologies, Carlsbad, CA) overnight at 4°C. The retinal flat mounts were mounted on slides using Prolong Gold anti-fade (Life Technologies). All images were taken with 4x magnification in a Cytation 5 cell imaging multimode reader microscope (BioTek, Winooski, VT).

RGCs were immunostained in whole-mounted retinas and in cryostat sections of the eye. The immunohistochemical techniques were performed at the same time for all species studied. The whole-mounted retinas were blocked with phosphate buffered saline, pH 7.4 (PBS) containing TX-100 (0.25%) overnight, prior to incubation in the primary antibody. The sections were washed twice with PBS-TX-100 for 10 min, and they were then incubated overnight with a primary guinea pig antibody or rabbit anti-RBPMS antibody (1:1000; Phosphosolutions, Aurora, CO, USA), and goat anti-Brn3a (1:1.000; Santa Cruz Biotechnology, Dallas, TX, USA) to detect RGCs, and primary mouse anti-Neurofilament 200 antibody (1:200; Vector Laboratories, Burlingame, CA, USA) to label neuronal axons. After two washes with PBS, antibody binding was detected for 1 h (5 h for whole-mount retinas) with Alexa Fluor 555 goat anti-guinea pig and Alexa Fluor 488 goat anti-mouse secondary antibodies, or Alexa Fluor 555 donkey anti-rabbit and Alexa Fluor 488 goat anti-goat secondary antibodies (Invitrogen, Carlsbad, CA, USA) diluted 1:1000 in PBS-BSA (1%). The sections were washed twice with PBS for 10 min and mounted with a coverslip in PBS:Glycerol (1:1).