Anti ph3 antibody

Pups were sacrificed at E14.5 and the embryonic brains were fixed, frozen, and cut into sagittal sections as previously described. An antigen retrieval step was performed by incubating the slides with 10mM Sodium Citrate buffer 10 min at 100 °C. Slides were then allowed to cool down and rinsed once with PBS before blocking 1h at room temperature with 10%NGS and 0.1% Triton X-100 in PBS. Anti-PH3 antibody (06-570, Merck Millipore) and anti-NeuN (ab177487, Abcam) were used alone while anti-PAX6 (901301, Biolegend) and anti-TBR2 (14-4875-80, ThermoFisher) antibodies were used concomitantly for co-staining. In all conditions, primary antibody was incubated overnight at 4 °C. After three washes with 0.1% Triton X-100 in PBS, slides were incubated with a matching secondary antibody (Alexa fluors, Thermo Fisher). Slides were then washed three times in PBS and mounted and scanned as previously described. We counted the PH3⁺ cells at the apical surface and in the SVZ independently in images of 650 μm of width and calculated the percentage of PH3⁺ cells at the SVZ. 250μm × 250 μm images were taken in the neocortex for the PAX6-TBR2 co-staining. Each cell type was counted independently. To analyze the NeuN labeling at E14.5, the thickness of the NeuN+ layer was measured at least four times along the neocortex.

For whole-mount immunostaining, chorions were removed by hatching enzyme for 20 min at room temperature. The samples were washed in PBS and were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 3 h. For immunohistochemical analyses, fixed embryos were incubated with polyclonal anti-phospho-histone H3 (Ser10) (anti-P-H3) antibody (1:200, Merck KGaA, Darmstadt, Germany), for 1 day at 4 °C, thereafter the samples were washed with PBS and further incubated with secondary antibodies conjugated with Alexa-488 (Invitrogen, Carlsbad, CA, USA, 1:1000) for 3 h for fluorescent images and were counterstained with DAPI for 1 hr. Fluorescent images were obtained using a confocal microscope (FluoView FV1000; Olympus, Tokyo, Japan). Photomicrographs of the samples were taken with a digital camera (DFC7000T, Leica Microsystems, Wetzlar, Germany) equipped with a fluorescence microscope (BX50, Olympus, Tokyo, Japan) and anti-P-H3-labeled cells were counted.