Comprehensive Mouse Brain Analysis

Mice were sacrificed at P14, P30 or 4 months by ketamine/ xylazine overdose followed by transcardial perfusion with saline solution (0.85% NaCl, 0.025% KCl, 0.02% NaHCO₃, pH 6.9, 0.01% heparin, body temperature) and ice cold 4% paraformaldehyde (PFA) freshly depolymerized in 1×phosphate-buffered saline (PBS), pH 7.4. The fixed brains were carefully isolated from the skull and were further stored overnight in the same ice-cold 4% PFA solution as used for transcardial perfusion. For further storage and cryoprotection, the brains were transferred to a mixture of 20% glycerol and 2% dimethylsulfoxide in 0.1 M phosphate buffer. Consecutive horizontal sections (40 μm) were collected in six series using a freezing microtome. Corresponding brain sections from wildtype (WT) and Ncald^KO/KO littermates (gender matched) were stained simultaneously for further immunohistochemical analysis as previously described (Kononenko et al., 2017 (link)). The following antibodies were used: anti-NCALD (1:100, 12925-1-AP, Proteintech), anti-NeuN (1:500, EPR 12763, Abcam), anti-TBR1 (1:500, ab31940), anti-CUX1 (1:200 sc-13024, Santa Cruz), anti-glial fibrillary acidic protein (anti-GFAP; 1:500, G3893, Sigma), anti-Ki-67 (1:500, ab15580 Abcam), anti-DCX (1:500, AB2253, Merck), anti-adenomatous polyposis coli (anti-APC; 1:500, OP80, Merck), anti-MBP (1:1,000 SMI94, Covance).