Anti aldh1a2

The western blot protocol was conducted as previously described [18 (link)]. The antibodies used in the study were as follows: anti-CD45 (BD Biosciences, 610266, (1/1000), anti-ROCK2 (Cell Signaling, 1/1000), SLC45A3 (Abcam, ab137065, 1/1000), and anti-ALDH1A2 (Abcam, ab156019, 1/2000). Unfortunately, there is no available specific antibodies for KCNK3 [19 (link), 20 (link)], thus avoiding any measurement of the protein expression. When tested in kcnk3-knockout miceand Kcnk3-mutated rats, we previously found that all commercially anti-KCNK3 antibodies tested were unusable [20 (link)]. This is why we analyzed KCNK3 expression by RT-qPCR. The blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse diluted 1:10,000 (Cell Signaling) or with HRP-conjugated goat anti-rabbit diluted 1:5000 (Cell Signaling). The antibodies were detected using ECL reagents. (Perkin–Elmer). ImageJ software was used to quantify the level of protein expression.

Tissues were fixed in 10% formalin, embedded in paraffin wax and cut into 6-μm sections. Immunostaining was carried out automatically using a BlueMap kit and the Discovery System (Roche) or manually for immunofluorescence staining. The following primary antibodies were used: anti-Sall126 (link) (PPMX Perseus Proteomics: PP-K9814-00); anti-Six2 (Proteintech); anti-Six2 (Abnova); anti-Wt1 (Santa Cruz); LTL (Vector); anti-Slc12a3 (Millipore); anti-cytokeratin (Sigma-Aldrich); anti-Ncam (Developmental Studies Hybridoma Bank #5B8); anti-E-cadherin (BD Biosciences: mouse-derived, Cell Signaling: rabbit derived), anti-CD31 (Abcam); anti-Pdgfrβ (Cell Signaling); anti-Aldh1a2 (Abcam); anti-PHH3 (Millipore; rabbit-derived, Abcam; mouse-derived); anti-Red Fluorescent Protein (RFP; Rockland); and anti-GFP (Abcam; chicken-derived). In paraffin sections, no GFP or tdTomato signals were detected unless the respective antibodies were used. TUNEL assays were performed using an ApopTag Plus fluorescein in situ apoptosis detection kit (Millipore), and the signal was enhanced using the biotin-conjugated anti-digoxigenin antibody (Sigma-Aldrich) and Alexa 594-conjugated streptavidin (Life Technologies). Immunofluorescence was visualised with an LSM780 confocal microscope (Zeiss).