Rabbit anti p smad2 3

Western blot analysis was conducted as previously reports [31 (link)]. The target protein was detected using primary antibodies as follows: Mouse anti-α-SMA (1:500, Sigma-Aldrich), Rabbit anti-p-Smad2/3 (1:1000, Cell Signaling Technology), Rabbit anti-Smad2/3 (1:1000, Cell Signaling Technology), Mouse anti-PPAR-α (1:1000, Abcam) and Mouse anti-β-actin (1:5000, Proteintech).

Embryos were incubated in ice-cold acetone (Roth, 5025.5) for 7 min, then washed at least three times with PBST, blocked for at least 1 h in 10% FBS in PBST and incubated in 1:5000 rabbit anti-pSmad2/3 (Cell Signaling Technology, 8828) in 10% FBS in PBST at 4°C overnight. Embryos were then washed at least five times in PBST, blocked again for at least 1 h in 10% FBS in PBST, and incubated in 1:500 goat anti-rabbit HRP secondary antibody (Jackson ImmunoResearch, 111-035-003) in 10% FBS in PBST at 4°C overnight. Next, embryos were washed at least five times in PBST, then once in TSA 1x amplification buffer (TSA Plus Cyanine 3 System, Perkin Elmer, NEL744001KT). For staining, embryos were incubated in 75 μl 1:75 Cy3-TSA in 1x amplification buffer in the dark at room temperature for 45 min. After washing at least six times with PBST, embryos were incubated in 1:5000 DAPI (Life Technologies, D1306; stock concentration: 5 mg/ml) in PBST at room temperature for at least 1 h, then washed at least four times with PBST. Finally, embryos were wrapped in aluminum foil and stored at 4°C overnight before SPIM imaging.

Primary antibodies used in this study were rabbit anti‐TSP50 (Abcam, ab181993), rat anti‐TSP50 (Novus, 260 514), rabbit anti‐β‐catenin (Abcam, ab223075), rabbit anti‐TGFβRI (Abcam, ab235578), rabbit anti‐TGFβRII (Abcam, ab259360) rabbit anti‐smad2/3 (Cell Signaling Technology, 8685), rabbit anti‐p‐Smad2/3 (Cell Signaling Technology, 8828), rabbit anti‐Spdef (LSBio, 211 521), rabbit anti‐Ki67 (ZSGB‐BIO, ZA‐0502). Secondary antibodies kits for immunohistochemical (IHC) and DAB staining kit were purchased from ZSGB‐BIO (Beijing, China). FITC‐conjugated goat anti‐rat IgG antibody, Cyanine3 (Cy3)‐conjugated goat anti‐rabbit IgG antibody for immunofluorescence (IF),4,6‐diamidino‐2‐phenylindole (DAPI) and X‐gal staining kit were purchased from Beyotime Institute of Biotechnology.
Hematoxylin and eosin (HE), Masson, Alcian Blue, Nuclear fast red staining kits and Carnoy's Fluid were purchased from Solarbio (Solarbio, Beijing, China). For organoid culture, Dulbecco's Modified Eagle Medium: F‐12 (DMEM/F12) medium, Mouse IntestiCult Organoid Growth Medium, Gentle Cell Dissociation Reagent, Dulbecco's Phosphate Buffered Saline (DPBS), and frozen stock solution were purchased from Stem cell Technologies(Vancouver, BC, Canada). Matrigel was from Corning (corning, NY, USA). 70‐µm cell strainers were from BD Bioscience (BD Bioscience, NA, USA).

Cultured cells and liver tissues were lysed using lysis buffer supplemented with protease and phosphatase inhibitors. Proteins (30 µg) were separated by SDS-PAGE. Rabbit anti-p-Smad2/3, T-Smad2/3, p-MEK, T-MEK, p-ERK, T-ERK, p-JNK, T-JNK, p-p38, T-p38, Caspase-8, Caspase-3, Caspase-9, and PARP were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti-α-SMA was purchased from Abcam (Cambridge, MA, USA). The immunoblotting signals were normalized to that for α-tubulin (Sigma-Aldrich, St. Louis, MO, USA). Sirius red staining (Abcam) of paraffin-embedded liver sections was used to qualitatively assess the collagen architecture and the extent of fibrosis in accordance with the manufacturer’s instructions.