Anti brn3a

Murine retinal sections embedded in low melting point agarose were prepared following our published methods (Nookala et al., 2010 (link)). Briefly, tissue sections were blocked with 10% goat serum and permeabilized with 2.5% Triton X-100. The following primary antibodies were used per manufacturers conditions: RBPMS (rabbit polyclonal IgG, Santa Cruz Biotechnology, 1:100 dilution); anti- γ-synuclein (SNCG, rabbit polyclonal IgG, GeneTex, 1:100 dilution); anti-BRN3A (goat polyclonal IgG, Santa Cruz Biotechnology, 1:10 dilution); anti-Neuronal Class III β-Tubulin (TUJ1, mouse monoclonal IgG2a, Covance, Princeton, NJ; 1:100 dilution); anti-HNK-1/N-CAM (CD57, Clone VC1.1, mouse monoclonal IgM, 1:10 Dilution); and CD15 (Clone:MC-480, mouse monoclonal IgM, BioLegend, 1:25 dilution). The appropriate Alexa Fluor-tagged secondary antibodies (Invitrogen, Waltham, MA; 1:200 dilution) and TO-PRO-3 iodide (Invitrogen; 1:4000 dilution) were used to indicate the location of the antigens of interest and nuclei, respectively. Sections were viewed and images were obtained using a Nikon C1 (Nikon, NY, USA) confocal microscope within the Imaging Core Facility in the Hamilton Eye Institute. All microscope settings, including laser levels and gain, were held constant to allow for relative comparisons of signal intensity within and between experiments.

Eye cups were dissected and immersed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 24 h and washed three times with PBS. Dissected eye cups were infiltrated with 30% sucrose overnight, embedded in OCT compound (International Medical Equipment) and frozen in isopentane on dry ice. Transverse sections of the retina (10 µm thick) were cut on a cryostat and mounted onto slides. Pre-IHC antigen retrieval was performed by incubating dried frozen sections for 20 min at 70°C with HistoVTOne (Nacalai USA). The frozen sections were then washed with PBS and blocked for at least 1 h in 20% goat serum in PBS. Primary antibodies used were anti-Brn3a (1:50; Santa Cruz Biotechnology, catalog number sc-8429) and anti-melanopsin (1:5000; Advanced Targeting Systems, catalog number AB-N38). Sections were incubated in primary antibodies overnight at 4°C and followed by three times washing with PBS. Primary antibody binding was detected by incubation with the following secondary antibodies, as appropriate, diluted 1:400 in PBS: Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 594 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 goat anti-mouse IgG. The sections were counterstained with DAPI (40, 60-diamidino-2-phenylinode hydrochloride) and coverslipped with Vectashield (Vector Labs). Sections were imaged with a Nikon Ti Microscope (Nikon).

Following perfusion (see above), eyes were enucleated, post-fixed in 4% paraformaldehyde for 2 hours and cryoprotected in 30% sucrose overnight. Eyes were then embedded in mounting medium (Tissue-Tek O.C.T Compound, Sakura Finetek Europe, Alphen aan den Rijn, Netherlands) and frozen. Twelve μm coronal sections were sliced for immunofluorescence. Sections were washed in PBS and blocked in 10% sera in 0.1% PBS-Triton X. Sections were incubated with anti Brn3a (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) and staining was visualized with the an Alexa 555-conjugated secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA). Sections were mounted in anti-fade medium containing 4′,6-Diamidin-2-phenylindol (DAPI) (Vector Laboratories).