Alexa 488 conjugated donkey antibody against rabbit igg

Histological analysis was followed by previous procedures (Mori and Morimoto, 2014 (link); Mori et al., 2019 (link)). Briefly, under deep anesthesia, 2-month-old male wild-type and SIK1-MT mice were perfused transcardially with ice-cold PBS (pH 7.4), followed by 4% paraformaldehyde in PBS. Fifty-micrometer-thick coronal sections were prepared with a sliding microtome (REM-700, Yamato Kohki Industrial). The sections were washed with PBS; blocked with PBS containing 1% bovine serum albumin, 0.1% Triton-X-100, and 10% of normal donkey serum; and incubated with mouse anti-parvalbumin (1:2000, Sigma), rat anti-somatostatin (1:200, Merck Millipore), or rabbit anti-Satb2 antibody (1:200, Abcam). After overnight incubation with primary antibodies, the brain sections were washed with PBS containing 0.1% Triton-X-100 and incubated with Alexa 488-conjugated donkey antibody against rabbit IgG or Cy2-conjugated donkey antibody against mouse or rat IgG (Jackson Immunoresearch), respectively, for 2–3 h at room temperature. After further washing with PBS, brain sections were mounted on a sliding glass, counterstained with DAPI, and coverslipped. Fluorescence images were taken with an all-in-one fluorescent microscope (BZ-X710, Keyence) and a confocal laser-scanning microscope (TCS SP8; Leica Microsystems).