Anti chat primary antibody

Immediately after dissection, spinal cord segments (2–3 mm) were fixed in 4% paraformaldehyde in PBS for 24 h as previously described [22] (link). After fixation, samples were cryoprotected by suspending them overnight in PBS containing 30% sucrose at 4°C and then sliced using a cryotome (30 µm). Free-floating tissue sections were made permeable (0.3% Triton X-100 in PBS) for 10 min and blocked in PBS blocking solution 1 (1% gelatin and 10% FBS) for 30 min at room temperature. Spinal cord sections (30 µm) were first incubated with an anti-ChAT primary antibody (24 h at 4°C, 1∶50, Millipore) and then revealed using a FITC goat anti-rabbit secondary antibody (2 h at room temperature, 1∶200, Jackson ImmunoResearch). Subsequently, sections were incubated with an anti-Ca_V3.1 primary antibody (24 h at 4°C, Santa Cruz Biotechnology; 1∶100 dilution), and then exposed 1 h to the secondary antibody (1∶200; Dylight 549-conjugated anti-rabbit IgG; Jackson ImmunoResearch). Samples were examined using confocal laser scanning microscopy (Leica TCS SP2). Images were obtained with the filter set for Dylight 549 using the 40× oil immersion plan apochromatic objective (NA 0.8).

Spinal cord sections of 30 µm were first incubated with an anti-ChAT primary antibody (24 h at 4°C, 1∶50, Millipore) and then revealed using a FITC donkey anti-goat secondary antibody (2 h at room temperature, 1∶200, Jackson ImmunoResearch). Subsequently, sections were incubated with an anti-α₆ subunit antibody (2 h at 4°C, Sigma; 1∶50 dilution), and then exposed 1 h to the secondary antibody (1∶200; Dylight-Jackson donkey; Jackson ImmunoResearch). Samples were examined using confocal laser scanning microscopy (Leica TCS SP2). Images were obtained with the filter set for Dylight 549 using the 20x and the 40x oil immersion plan apochromatic objective (NA 0.8).