Tsc 5sp x confocal microscope

E10.5 embryos were fixed in 4% (w/v) paraformaldehyde (PFA) in PBS at 4°C. Embryos were then immersed in 15% and 30% sucrose and embedded in Tissue Freezing Medium (Triangle Biomedical Sciences, Inc.). Sections were taken at 8 μm. For immunostaining sections were blocked in 10% donkey serum/1% BSA and permeabilized in 0.1% Tween-20. Primary antibodies are listed in Supplementary Table 1. Secondary antibodies were: donkey anti-mouse Alexa Fluor 488, donkey anti-mouse Alexa Fluor 594, donkey anti-rabbit Alexa Fluor 488, donkey anti-rabbit Alexa Fluor 594 (Molecular Probes), goat anti-chicken Alexa Fluor 488 (Molecular Probes), and donkey anti-rabbit Alexa Fluor 647 (Molecular Probes). Samples were mounted in ProlongGold (Molecular Probes) and images captured using a Leica DM2500 optical microscope or Leica TSC 5SP X confocal microscope (Leica Microsystems). EdU staining on paraffin embedded kidney sections was conducted using Click-iT™ EdU imaging kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Animals were injected EdU at 25 mg/kg and euthanized 2 hours later for analysis. A least 3 individual kidneys were analyzed per genotype per age group. From each kidney 10 images from the cortex were taken at 200x magnification, and the total number of EdU+ nuclei and DAPI+ nuclei in the DBA+ collecting ducts were counted for calculating the EdU ratio.

Primary antibodies are listed in Supplementary Table 1. Secondary antibodies were: donkey anti-mouse Alexa Fluor 488, donkey anti-mouse Alexa Fluor 594, donkey anti-rabbit Alexa Fluor 488, donkey anti-rabbit Alexa Fluor 594 (Molecular Probes), and goat anti-chicken Alexa Fluor 488 (Molecular Probes). Samples were mounted in ProlongGold (Molecular Probes) and images captured on a Leica TSC 5SP X confocal microscope (Leica Microsystems).