Cd31 antibody

After overnight fixation in 4% paraformaldehyde fixative, the kidneys of newborn mice were dehydrated and embedded in paraffin for CD31 staining. Slides with 5 micron sections were stained at the Memorial Sloan-Kettering Cancer Center Molecular Cytology Core Facility with Ventana staining processors (Ventana, Roche), using standard protocols with CD31 antibody (1 μg/ml, from Dianova, cat. # DIA-310). Stained slides were analyzed in a blinded manner. Several representative stages of kidney development (i.e. S-shaped bodies, maturing glomeruli, and mature glomeruli) were identified and imaged per mouse. Those images were then again compared in a blinded manner. A total of six newborn mice were analyzed (3 pairs of littermates; each pair with one control and one mutant animal). The PLVAP staining was performed on frozen sections prepared from six mutant and littermate control mice (4 females and 2 males) that were 6 weeks old, and the bound secondary alkaline-phosphatase (AP) labeled antibody was visualized by incubation with the AP substrates BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitro blue tetrazolium). NBT-BCIP (Roche) staining in NTMT (100mM NaCl, 100mM Tris-HCl pH9.5, 50mM MgCl2, 1% Tween20, 20μM Levamisole, in H2O) occurred for 2 hours at room temperature.