Rabbit anti caspase 3

Heterozygous dams at embryonic day 12.5 (E12.5), day 14.5 (E14.5) and day 16.5 (E16.5) were injected with a single dose of BrdU solution (100 mg/kg). Pregnant dams were killed at day 18.5 of gestation, and embryos were harvested. Embryo brains were drop fixed in 4 % PFA for 12 h at 4 °C then cryopreserved in 20 % sucrose solution. WT and Brinp1^−/− embryo brains were cryopreserved in OCT medium and sectioned to 14 μm. For immunostaining of BrdU, slides were incubated in 1:7 HCL: PBS (37 % concentrated stock) at 37 °C for 1 h. Slides were then washed in PBS and permeabilized with PBS-0.1 % Triton X-100, then blocked with 10 % NGS/PBS-Triton for 30 min. A BrdU antibody (1:100, BD) was applied at RT for 2 h and counter stained with DAPI. Sections were also stained with the following antibodies: rabbit anti-Ki67 (1:1000, Leica), rabbit anti-pHH3 (1:400, Millipore) and rabbit anti-caspase 3 (1:1000, R&D systems).
Images for all immunostaining were obtained using a Nikon C1 confocal microscope. Cell count analysis were performed blind of genotype using Imaris 7.6.3. A minimum of three representative sections per mouse were analysed by dividing cortical regions into equal bins. Repeat measures two-way ANOVAs were performed for comparisons between WT and knock-out tissue. Statistical tests were performed with GraphPad Prism 5 (GraphPad).

Brainstem slides were baked at 60°C for 30 min, de-waxed and rehydrated. Antigen retrieval was performed in citrate buffer (pH 6) using a microwave for 15 min. Endogenous peroxide quenching was performed by incubating slides in 0.1% H₂O₂ in methanol. 3% NGS in 1 × PBS was used to prevent non-specific binding. Sections were labelled with 1:800 rabbit-anti-Caspase 3 (R&D systems, cat#: AF835) in 3% NGS and incubated overnight at 4°C. Slides were then incubated in biotin conjugated IgG [1:200 goat anti-rabbit (DAKO, Victoria, Australia)] secondary antibody for 3 h at room temperature before being incubated in avidin-biotin complex (Sigma-Aldrich) for 45 min. Sections were reacted with 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich). Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was used to identify cells undergoing in-situ apoptosis using the ApopTag® Peroxidase Kit as per manufacturer's instructions (Millipore S7100; CA, USA). Brainstem sections stained with both caspase-3 and ApopTag were then incubated in anti-digoxigenin conjugate for 30 min at room temperature, before being incubated with diaminobenzidine peroxidase substrate. PBS was used to stop the reaction. Slides were then dehydrated, and cover slipped.