Publication protocol
Embryos were fixed in buffered 3.7% paraformaldehyde and equilibrated to 30% sucrose. Cryosectioning of Neg50 (Fisher Scientific/Thermo Scientific, Pittsburgh, PA) embedded tissue was cut with a Leica HM550 cryostat and immunolabeling completed as described (Darland et al., 2011). In brief, sections were blocked and permeabilized in 3% donkey serum, 2% goat serum (Vector Laboratories, Burlingame, CA), 0.1% Triton X-100, and 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) overnight at 4°C. The primary antibody incubation was 2 hours at room temperature or overnight at 4°C. The absence of primary antibody or use of species-matched immunoglobulins were used as negative controls. Rabbit polyclonal antibodies were phospho-histone H3 (pHH3, 1:200, Upstate Biotechnology, Lake Placid, NY), Tbr2, and Ctip2 (1:400, Abcam, Cambridge, MA). For colormetric detection, biotinylated secondary antibodies (Goat anti-rabbit~biotin, 1:200; Jackson Laboratories) were incubated for 45 minutes at room temperature, followed by Vectastain Elite ABC horseradish peroxidase staining as per the manufacturer’s protocol. The immunolabeled sections were counterstained with methyl green. For immunofluorscent detection of primary antibodies, fluorochrome-coupled secondary antibodies were incubated in block solution for one hour at room temperature (cy3, 1:200; Jackson Immunoresearch Laboratories, West Grove, PA). Nuclei were labeled with DAPI (Sigma Chemical Company, St. Louis, MO).
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Manufacturer protocol
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