Anti chx10

The immunohistochemical methods have been described previously.²⁸ (link)^,31 (link)^,32 (link) Eyecups were fixed with 4% paraformaldehyde, cryoprotected, and embedded in tissue freezing medium (Electron Microscopy Sciences, Hatfield, PA, USA), and 10- to 15-µm-thick frozen sections were cut. The tissues were incubated with diluted primary antibodies overnight (sections) or for 48 hours (whole mounts), washed, and then incubated in secondary antibodies for 2 to 4 hours. Images of immunolabeled retinal sections or whole mounts from control and experimental groups were obtained with an Olympus FV1200 MPE confocal microscope (Olympus, Tokyo, Japan) using a 40× oil-immersion objective. High-resolution (1024 × 1024 pixels) z-stack images were taken using step sizes of 0.7 to 2.0 µm and compiled. The brightness and contrast of micrographs were adjusted using Photoshop CS6 (Adobe, San Jose, CA, USA). The primary antibodies were anti-PKCα (1:10,000; Sigma-Aldrich); anti-Chx10 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA); anti-calbindin (1:500; Santa Cruz Biotechnology); anti-SMI32 (1:2000; Covance, Princeton, NJ, USA); anti-VGlut1 (1:150; BioLegend, San Diego, CA, USA); and anti-CtBP2 (1:500, Santa Cruz Biotechnology). Secondary antibodies were conjugated with Alexa Fluor 488, 594, and 633 (1:200; Thermo Fisher Scientific, Waltham, MA, USA).