Rabbit anti phospho histone h3 antibody

For cell proliferation, 4-μm sections of paraffin-embedded kidneys were incubated with a rabbit anti-phospho-histone H3 antibody (Millipore) at 1:200 and a mouse anti-PCNA antibody (DAKO) at 1:50, followed by the appropriate secondary antibody. The number of proliferating cells was determined in UB and expressed as the number of pH3- or PCNA- positive cells per UB structure on whole kidney sections.
Apoptosis was detected in 4-μm sections of paraffin-embedded kidneys by TUNEL assay using the In Situ Cell Death Detection kit (Roche) according to the manufacturer’s protocol. The number of apoptotic cells was determined as the number of TUNEL–positive nuclei per microscopic field in whole kidney sections. Ten microscopic fields were scored for each kidney in 5 embryos of each genotype.

Brain sections were prepared as described for in situ hybridization. The sections were washed with TBST (10 mM Tris pH7.4, 150 mM NaCl, 0.1% Tween20), permeablized with 100% methanol at -30°C for 15 min, and then reacted with the primary antibodies at 4°C overnight. The following antibodies were used: mouse anti-Cadherin7 antibody [CCD7-1; Developmental Studies Hybridoma Bank (DSHB), Iowa, IA, USA], rat anti-Ctip2 antibody (ab18465; Abcam, Cambridge, UK), rabbit anti-Foxp1 antibody (ab16645; Abcam), mouse anti-β (III)-tubulin antibody, TUJ1 (MMS-435P; Covance, Princeton, NJ, USA), and rabbit anti-phospho-Histone H3 antibody (06-570; Millipore, Billerica, MA, USA), After washing with TBST, the sections were stained with anti-mouse IgG antibody conjugated with Alexa Fluor 488 (A11029; Life Technologies), anti-rat IgG antibody conjugated with Alexa Fluor 488 (A11006; Life Technologies) or anti-rabbit IgG antibody conjugated with Cy3 (711-165-152; Jackson ImmunoResearch, West Grove, PA, USA). They were then washed with TBST again, and coverslipped with 90% glycerol in PBS.