Ab2253

DNA was denatured with 2N HCl during 10 min at 37°C followed by 10 min in boric acid 0.1 M (pH 8.5) at room temperature. The tissues were rinsed in PBS-Triton-X-100 0.03% three times for 5 min, and blocked with goat serum 10%. They were incubated with primary antibodies for 20 h at 4°C: rat anti–BrdU (1:400; AbD Serotec Cat# OBT0030 RRID: AB609568) plus mouse anti-Nestin (1:400; Millipore Cat# MAB353 RRID:AB94911), guinea pig anti-DCX (1:500, Millipore Cat# AB2253 RRID:AB1586992), or mouse anti-NeuN (1:400; Merck Cat# MAB377 RRID:AB11210778). After rinsing, the sections were incubated with appropriate secondary antibodies (1:1000, Alexa Fluor 488 for BrdU, Alexa Fluor 594 for cell markers; Invitrogen, Life technologies) for 60 min at room temperature. The brain tissues were mounted with fluorescence solution Vecta Mount (Vector, H-5000) and quantified under a fluorescence microscope Axio-observer D2 (Zeiss, Germany).

EBs (6, 8, and 12 days old) were fixed with 1% paraformaldehyde during 15 min at room temperature (RT). Fixed EBs were cryoprotected in 15% sucrose in PBS, embedded in a solution containing 7.5% gelatine (Sigma-Aldrich) and 15% sucrose in PBS, frozen and cryosectioned (8–10 μm). EB sections were immersed in PBS at 37°C until gelatine was completely dissolved, and then processed for immunocytochemistry. Sections were blocked with 10% FBS and 0.05% Tween in PBS for 1 h, followed by incubation overnight with the following primary antibodies: Anti-MyoVIIa (1:400, HPA028918, Sigma), Anti-MyoVI (1:50, 25-6791, Proteus Biosciences) Anti-Pou4f3 (1:50, HPA038215, Sigma), Anti-Espin (1:1,000, gift of A. J. Hudspeth), Anti-Gfi1 (1:2,000, gift of Hugo Bellen), Anti-Gfi1 (ab21061 or ab290, Abcam), Anti-Lhx3 (1:100, ab14555, Abcam), Anti-Tuj1 (1:500, MMS-435P, Covance), Anti-GFP (1:500, ab13970, Abcam), Anti-Doublecortin (1:1,000, AB2253, Merck Millipore). Sections were washed 3 times in PBS followed by incubation for 1 h at RT with AlexaFluor-conjugated secondary antibodies (1:400, Molecular Probes) and 0.15% DAPI (Sigma-Aldrich). Slides where then mounted with prolong gold (Life technologies). Fluorescent images of fixed sections were captured with Widefield Zeiss observer or using a Leica TCS SP8 Confocal 4 Detectors. All digital images were formatted with Adobe Photoshop CS and ImageJ.

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).