Retinas from wt mice were dissected and fixed in 4% PFA for 20 min. After fixation, retinas were infiltrated with graded (10%, 20%, and 30%) sucrose in PB and sectioned vertically at 14 μm on a cryostat (Leica). Immunohistochemical labeling was conducted by using the indirect fluorescence method. Retinal sections were blocked in 1% bovine serum albumin (BSA) in 0.1% Triton X-100 in PBS (PBST) for 2 h. Following removal of the blocking solutions, retinal sections were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: rabbit anti-CaM (1:500, #301003, Synaptic Systems), mouse anti-PKCα (1:200, #P5704, Sigma), and mouse anti-CtBP2 (1:500, #612004, BD Biosciences). After rinsing, corresponding secondary antibodies including Alexa Fluor 488 donkey anti-rabbit (1:200, #A21206, Thermo Fisher Scientific) and Alexa Fluor 568 donkey anti-mouse (1:200, #A10037, Thermo Fisher Scientific) were applied for 2 h in darkness. All antibodies were diluted with 5% BSA in PBST. After rinsing, DAPI stain (1:1000, #C1002, Beyotime) was applied for 10 min at room temperature. Between incubations, sections were washed three times for 5 min each using PBST. Control experiments were conducted either by preincubation of the anti-CaM antibody with the CaM or Ca2+-binding protein 5 (CaBP5) immunopeptides (EEFVQMMTAK corresponding to AA 140–149 in rat/mouse CaM, #301-0P, Synaptic Systems; EEFVKMMSR corresponding to AA 165–173 in mouse CaBP5, synthesized by Sangon Biotech) or by omission of both primary antibodies. All labeled sections were examined with a confocal laser scanning microscope (LSM 880, Carl Zeiss) with Plan-Apochromat 63×/1.4 or 100×/1.40 oil-immersion objectives. Images were adjusted for contrast and brightness by using ZEN software (Carl Zeiss) or Photoshop software (Adobe Systems).

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