Mouse anti ctbp2 612044

Generation of rabbit anti-RGS11^{106 (link)}, sheep anti-TRPM1, sheep anti-mGluR6 and rabbit anti-Elfn1 antibodies were described previously ^{68 (link),96 (link)}. Rabbit anti-Ca_V1.4 ^{73 (link)} was a generous gift from Dr. Amy Lee (University of Iowa, IA). Rabbit anti-LRIT3^{107 (link)} was a generous gift from Dr. Christina Zeits (Institut de la Vision). Commercial antibodies were: mouse anti-CtBP2 (612044, BD Biosciences), mouse anti-PKCα (ab11723; Abcam), rabbit anti-PSD95 (D27E11, Cat# 3450S, Cell Signaling Technology).
Adeno associated virus (AAV8)- based vector containing three surface-exposed proteosomal avoidance mutations (Y447F, Y733F and T494V) was packaged with a construct containing the human rhodopsin kinase 1 (hGRK1) promoter driving Cre recombinase, followed by a P2A cleavage site and green fluorescent protein (GFP)(pTR-hGRK1-Cre-P2A-GFP). Control vectors (packaged in the same capsid) contained hGRK1 driving GFP. Use of the hGRK1 promoter ensures photoreceptor specificity for Cre recombinase and GFP expression ^{108 (link)}.

PER2, wild type or mPer2^Brdm1 mutants (having a frame deletion that produces an unstable PER2 protein) [35 (link)] underwent transcardiac perfusion (4% paraformaldehyde) and decalcified in EDTA 2% for 4 days. Cryosections were immunostained with a rabbit-antibody to PER2 (PER21-A, Alpha Diagnostic, Texas USA; 1:100). For the quantification of synaptic ribbons, surface preparations were stained for C-terminal binding protein 2 (mouse anti-CtBP2, 612044 from BD-Biosciences, used at 1:200) and secondary FITC-conjugated goat anti-rabbit and TRIC-conjugated goat anti-mouse antibodies (Jackson ImmunoResearch; Pennsylvania USA; 1:100). Confocal image stacks were analyzed using Image J software. Quantification was performed using an automated particle counting after converting the image to grayscale and thresholding the image. This technique was manually validated before collecting the data.