Mouse anti calbindin cb

We perfused mice transcardially with PBS followed by 4% paraformaldehyde, and soaked the brains in 30% sucrose. We sectioned OBs coronally (30 µm) on a sliding microtome and performed immunohistochemsitry on floating sections in 5% heat-inactivated goat serum and 0.5% triton in PBS. We used the following primary antibodies: rabbit anti-calretinin (CR) (1:2000) and mouse anti-calbindin (CB) (1:1000) (Swant, Bellinzona, Switzerland), mouse anti-Tyrosine Hydroxylase (TH) (1:500) (Immunostar, Hudson, WI, USA), mouse anti-Tbr2 (1:100) (Abcam, Bristol, UK) and mouse anti-NeuN (Millipore, Billerica, MA, USA). GCaMP was amplified using chicken anti-GFP (1:1000, Millipore). The following secondary antibodies were used: biotinylated goat anti-rabbit (1:500), DyLight549 conjugated goat anti-mouse (1:500) and DyLight488 conjugated goat anti-chicken, (Jackson ImmunoResearch, West Grove, PA, USA). Amplification was carried out using Cy5 conjugated streptavidin (Jackson ImmunoResearch). Slices were imaged with a SP50 confocal microscope, via a 60X (1.4 NA) oil objective (Leica, Wetzlar, Germany). Counting of neuronal somata was carried out manually using ImageJ.

Free-floating fixed sections were blocked in 5% goat serum, 0.5% Triton X-100 (TX) in PBS for 1 h at room temperature, and then were incubated with primary antibody in 1.25% goat serum and 0.125% TX in PBS for 48 h at 4°C. Sections were then washed 3× with PBS and incubated with secondary antibodies in 1% goat serum and 0.1% TX in PBS for 2 h at room temperature. Finally, sections were washed 3× with PBS and mounted in Vectashield Antifade (Vector Laboratories) or Prolong Diamond (Thermo Fisher Scientific) mounting medium. Sections stained with mouse primary antibodies were blocked in ReadyProbes “Mouse on Mouse” IgG blocking solution (Thermo Fisher Scientific) for 1 h before the initial blocking step. Primary antibodies and dilutions used were rabbit anti-calretinin (CR; 1:2000; Swant), mouse anti-calbindin (CB; 1:500; Swant), and rabbit anti-neuropeptide Y (NPY; 1:2000; Immunostar). Secondary antibodies used were Alexa Fluor 647 anti-rabbit (1:1000; Thermo Fisher Scientific) and Alexa Fluor 647 anti-mouse (1:500; Thermo Fisher Scientific).