Rabbit anti cone arrestin

Mouse eyes were fixed in 2% PFA at room temperature for 2 h. The retinas were dissected as previously described [24 (link)]. Samples were incubated with 1% Triton X-100 in PBS for 4 h, and blocked by incubation with 1% BSA. Retinal flatmounts were incubated with rabbit anti-cone arrestin (Millipore, Watford, UK) at 4 °C overnight, followed by Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Life Technologies Ltd., Paisley, UK) for 2 h. Images were obtained by confocal microscopy (Eclipse TE200-U, Nikon UK Ltd., Surry, UK). The Fiji Image-J Software was used for image analysis.

Rabbit anti-calbindin (horizontal cells; Swant; CB38a; 1:1,000), Rabbit anti-cone arrestin (cones; Millipore; AB15282; 1:5,000), rabbit anti-PSD95 (synapses; Cell Signaling Technology; 1:1000), mouse anti-CTBP2 (synapses: BD Biosciences 1:1,000), peanut lectin (cone terminals: Millipore 1:2,000). Goat anti Bassoon (synaptic stains; Santa Cruz Biotechnology, Santa Cruz, CA; sc-18565; 1:400), and DAPI reagent (mixed into the second wash after incubation with secondary antibodies at a dilution of 1:50,000 of a 1 mg/ml stock). Secondary antibodies were acquired from Jackson Immuno Research and used at a concentration of 1:1000. Tissue was stained as previously described [40 (link)]. Briefly, antibodies were diluted in a blocking solution of 0.1% triton (sections) or 0.4% triton (whole retinas) supplemented with 7% normal donkey serum in PBS. Sections were blocked for 20 minutes in blocking solution and then incubated in primary antibody overnight at 4° C or at room temperature for one hour. Whole retinas were blocked for two hours in blocking solution incubated at 4° C for four days. Sections were washed 3 x for five minutes in PBS, while the whole retina washes were carried out for one hour. Tissue was incubated in secondary antibody diluted in blocking solution, as performed for primary antibodies, washed and mounted in 80% glycerol.