Immunohistochemistry Protocol for Retinal Cell Markers

Immunohistochemistry was conducted as previously described (Coomer and Morris, 2018 (link)) and imaged on a confocal microscope (Leica SP8, Leica). The following antibodies were used: anti-zCDHR1a (CDHR1a, rabbit, 1:100, Bosterbio, Pleasonton, CA, United States), anti-zSiah1 (Siah1, rabbit, 1:100, Bosterbio, Pleasonton, CA, United States), anti-Huc/D (ganglion and amacrine cells, mouse, 1:40), anti-PKCα (bipolar cells, mouse, 1:100, Santa Cruz Biotechnology, Dallas, TX, United States), anti-Prox1 (horizontal cells, rabbit, 1:1000, Acris, San Diego, CA, United States), anti-PCNA (cells in S-phase, mouse, 1:100, Santa Cruz Biotechnology, Dallas, TX, United States), and activated caspase 3 (apoptotic cells). Alexa fluor conjugated secondary antibodies (Invitrogen, Grand Island, NY, United States) and Cy-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used at 1:200 dilution and DAPI to label nuclei (1:10,000, Sigma, St. Louis, MO, United States). TUNEL assay was conducted with ApopTag Fluorescin Direct In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, United States) on retinal cryosections according to manufacturer’s instructions.

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Piedade W.P., Titialii-Torres K., Morris A.C, & Famulski J.K. (2020). Proteasome-Mediated Regulation of Cdhr1a by Siah1 Modulates Photoreceptor Development and Survival in Zebrafish. Frontiers in Cell and Developmental Biology, 8, 594290.

Publication 2020

anti-PKCα anti-PCNA Alexa fluor conjugated secondary antibodies Cy-conjugated secondary antibodies

Amacrine cells Antibodies Apoptosis Assay Caspase 3 Cells Confocal microscope Cryosections Dapi Dilution Ganglion Immunohistochemistry Mouse Nuclei Pcna Pkcα Rabbit Retinal Tunel

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Other organizations : University of Kentucky

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