Rabbit anti sox9

Mouse ureters were fixed in 4% paraformaldehyde for 30 min and embedded in Optimal Cutting Temperature compound (Tissue-Tek). Frozen samples for immunohistochemistry were cut into 6 µm in thickness. Primary antibodies used in this study were as follows: rabbit anti-Smad4 (1∶100, Millipore), rabbit anti-Sox9 (1∶300, Millipore), mouse anti-αSMA (1∶100, Sigma), rabbit anti-SM-MHC (1∶100, Biomedical Technologies), rabbit anti-Uroplakin (1∶100, a generous gift from Dr. Tung-Tien Sun, NYU) [22] (link). Alexa Flour 488 or 594 conjugated secondary antibodies (1∶500; Invitrogen) were applied to detect the corresponding primary antibodies. Section RNA in situ hybridization was carried out on 12-µm cryosections with methods described previously [23] , [24] (link).

For cryosections, embryos were fixed in 4% paraformaldehyde (PFA), cryoprotected in 30% sucrose in phosphate-buffered saline (PBS) overnight at 4°C, embedded in OCT compound (Tissue Tec; Sakura Fine Technical, Japan), and sectioned at 10 μm using a cryostat (HM560; Thermo Fisher Scientific, Waltham, MA). Specimens were blocked with horse serum for 1 hr at room temperature and incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1 hr at room temperature.
For flat-mount immunostaining of the RPE/choroid, the tissues were fixed with 4% PFA at room temperature for 30 min, and washed three times with PBS containing 0.5% Triton X-100 (PBST, Nacalai Tesque, Japan), incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1 hr at room temperature (Zhu et al., 2012 (link)). The primary antibodies and dilutions used were as follows: goat anti-Aldh1a1 (1:1,000; Abcam), rabbit anti-Aldh1a3 (1:1,000; Sigma), rat anti-endomucin (1:400; Millipore), mouse anti-GS (1:1,000; Millipore), FITC-isolectin B4 (1:100; Vector Laboratories), rabbit anti-ZO-1 (1:100; Invitrogen), rabbit anti-GFP (1:1,000; Abcam), rabbit anti-ERG (1:400; Abcam), rabbit anti-Pax6 (1:200; BioLegend), and rabbit anti-Sox9 (1:200; Millipore).

The paraffin sections were dewaxed and rehydrated. For cryosection, tissues were fixed in 4% PFA overnight before immersed in 30% sucrose. Sections were blocked with blocking buffer (5% BSA or goat serum, 0.5% Tween20) for 1 hour at room temperature. The primary antibodies of rabbit anti-Foxp2 (1:400; Abcam), rabbit anti-SOX9 (1:500, Millipore), guinea pig anti-SOX9 (1:2000, gift from V. Lee, STEMCELL Technologies) and rabbit anti-FOXA2 (1:500, Millipore) were diluted in blocking buffer and applied on the sections at 4°C overnight. The signal was visualized by using 1:500 goat-anti-rabbit or donkey-anti-guinea pig antibodies and mounting with Vectashield® mounting medium containing DAPI.

Lung explant cultures were fixed in MEMFA (3.8% formaldehyde, 0.15 M MOPS, 2 mM EGTA, 1 mM MgSO₄, pH 7.4) and permeabilised with 1% Triton-X 100 (Sigma-Aldrich, Poole, UK). Isolated epithelial cultures were first removed from culture using Matrigel recovery solution (BD Bioscience) before being fixed with 4% paraformaldehyde and permeabilised with 0.05% saponin.
Samples were then blocked with 2% BSA in PBS and incubated with either mouse anti-E-Cadherin (Cat No: 610182; BD Bioscience), rabbit anti-SOX9 (Cat No: AB5535; Millipore, Watford, UK), mouse anti-Ki67 (Cat No: 610968; BD Bioscience), rabbit anti-phospho-histone H3 (PH 3; Cat No: 05-806; Millipore), rabbit anti-TTF1 (Nkx2.1; Cat No: WRAB-1231; Seven Hills, Cincinnati, USA) or rabbit anti-pro-surfactant protein C (SPC; Cat No: WRAB-9337; Seven Hills). Samples were then incubated with either FITC conjugated horse anti-mouse (Vector labs, Birmingham, UK) or Alexa Flour-568 donkey anti-Rabbit (Invitrogen, Paisley, UK) secondary antibodies prior to mounting. Where indicated, Nuclei and F-actin were visualised by incubating the samples with DAPI and rhodamine-conjugated phalloidin (Sigma-Aldrich) respectively prior to mounting.
Fluorescent images were acquired using a Zeiss LSM 510 confocal microscope.

Primary antibodies used in this study included: mouse anti-BrdU and mouse anti-β-catenin, BD Biosciences (San Jose, CA); rabbit anti-phospho-S6 ribosomal protein (Ser240/244) XP mAb, rabbit anti-phospho-mTOR (Ser2448) mAb, Cell Signaling (Danvers, MA); mouse anti- β-actin, Sigma-Aldrich (St. Louis, MO); rabbit anti-Sox9, Millipore (Bilerica, MA) and mouse anti-active β-catenin Milipore (Bilerica, MA). The secondary antibodies used were HRP-conjugated anti-rabbit IgG, Cell Signaling and HRP-conjugated anti-mouse IgG, Thermo Scientific (Cincinnati, OH). Dulbecco's Modifid Eagle Medium (DMEM), M199 and cosmic calf serum were from HyClone (Logan, UT). Tamoxifen, Alcian blue, human apo-transferin, hydrocortisone and sodium selenite were obtained from Sigma-Aldrich. Human epithelial growth factor was from Peprotech (Rocky Hill, NJ). Human insulin and 0.05% Trypsin-EDTA were from Invitrogen (Carlsbad, CA). Rapamycin (RAP) was purchased from LC Laboratories (Woburn, MA).

Cell extracts were prepared as previously described [65 (link)] and analysed using the following primary antibodies: rabbit anti-Sox9 (Millipore, AB5535), rabbit anti-ALDH1A3 (Abgent, RB16818), mouse anti-GAPDH (Sigma, G8795), mouse anti-β-actin (AC-15/A5441), mouse anti-ERα (Novocastra Leica Biosystems, NCL-ER-6F11), Armenian hamster anti-Muc-1 (ThermoFisher Scientific, MA5-11202), mouse anti-β-tubulin (Sigma, T4026) and rabbit anti-Hsp60 (Santa Cruz Biotechnology, sc-13966). For detection an enhanced chemiluminescence detection kit (Bio-rad) was used.