Rabbit anti six2

Samples were blocked at least overnight in 5% normal donkey serum, 5% normal goat serum in TBS + 0.1% Triton-X, or PBS + 0.1% Triton-X prior to treatment in primary antibody diluted in blocking solution. Washes and antibody incubations were performed in TBS + 0.1% Triton-X or PBS + 0.1% Triton-X at 4°C for at least 24 hr each step. Antibodies used were Rabbit anti-SIX2 (Proteintech, 11562–1-AP), Mouse anti-SIX2 (clone 3D7, Abnova, H00010736-M01), Rabbit anti-RFP (MBL, PM005), Chicken anti-GFP (Abcam, ab13970), Rabbit anti-JAG1 (Cell Signalling, 2620), Rabbit anti-aPKC-zeta (Santa Cruz, sc-216), Rat anti-NCAM (GeneTex, GTX19782). Secondary antibodies labelled with Alexa-488, Alexa-568, Alexa-647 or BV-421 were purchased from Thermo Fisher or Jackson ImmunoResearch. In some experiments nuclei were labelled with Draq5 (Abcam, ab108410). Samples for Wnt4 lineage analysis were cleared using the previously described BABB based method (Combes et al., 2014 (link)). Constitutive Wnt4-Cre-labelled samples were cleared with Ethyl Cinnamate (Klingberg et al., 2017 (link)). Samples to identify native tdTomato signal alongside aPKC and NCAM were cleared using passive clarity (Yang et al., 2014 (link)). Samples were mounted in 35 mm glass bottom dishes (MatTek) for imaging.

Whole kidneys were fixed in 4% PFA for half an hour at 4 °C then placed in PBS. Whole mount immunofluorescence, confocal microscopy, and OPT was carried out according to published protocols, briefly, after initial in vivo cell labeling with the nucleoside analog 5-ethynyl-2’-deoxyuridine (EdU) and tissue-specific antibodies, OPT and confocal microscopy are used to image the developing kidney. These imaging data then inform a second analysis phase that quantifies (using Imaris and Tree Surveyor software), models and integrates these events at a cell and tissue level in 3D space and across developmental time. Cell counts per niche (confocal) and niche counts (OPT) were performed as reported^{36 (link)}. Antibodies used: rabbit anti-Six2 (1:600, Proteintech Group, #11562-1-AP), anti-rabbit Alexa Fluor-568 conjugated secondary antibody (1:300, Life Technologies).