Comprehensive Kidney Development Analysis

Histology, immunohistochemistry, and in situ hybridization were carried out according to standard procedures. Briefly, kidneys were fixed in 4% paraformaldehyde (PFA) for overnight at 4°C, dehydrated, and embedded in wax or OCT. Paraffin or frozen sections were generated at 6 μm of thickness. We used six embryos for each genotype at each stage for each probe and the result was consistent in each embryo. Probes for ISH were reported previously (Xu et al., 2014 (link)). Cy3-, Cy2-, Cy5-and FITC-conjugated secondary antibodies were used and Hoechst was used for nuclear counter-staining.
Primary antibodies: Anti-Six1 (12,891, Cell Signaling), -Six2 (MBS610128, MyBiosource), anti-Wt1 (sc-192, Santa Cruz Biotechnology), -Eya1 (25-067, Prosci Inc. and MABE1047, Sigma), and -PH3 (ab10543, Abcam).