Cells cultured on 6-well plates (1000 μL/well)/12-well plates (400 μL/well) were divided into different groups. After washing with phosphate-buffered saline + 0.05% Tween (PBST) three times (5 min each time), the cells were fixed with precooled methyl alcohol for 20 min at − 20 °C. Triton X-100 (0.5%) was added to the cell slide and incubated for 20 min for transparency. After that, the cells were washed with PBST and blocked with bovine serum albumin (BSA) (Abcone) for 30 min. Finally, the cells were incubated overnight with SOX17 (Rabbit, Proteintech, 1:100)/FOXA2 (Mouse, Abcam, 1:200), HNF4A (Rabbit, Abcam, 1:200)/AFP (Mouse, ABclonal, 1:100), CK19 (Rabbit, Proteintech, 1:100)/ALB (Mouse, Proteintech, 1:100), or β-catenin antibody (Rabbit, Proteintech, 1:100) at 4 °C. After washing 3 times, the cells were incubated with goat anti-mouse/rabbit IgG-FITC or goat anti-mouse/rabbit IgG-RBITC fluorescent secondary antibodies (Earthox, 1:500) for 60 min. Then, the cells were mixed with DAPI reagent for 10 min. Cell slides were observed under an optical microscope (MF52-N, Mshot) or with Axio Imager M2 software (Nikon) (×100, ×200 magnification).
β catenin antibody
Manufactured by Proteintech
Sourced in United States, China
The β-catenin antibody is a laboratory reagent used for the detection and analysis of β-catenin protein expression in various experimental systems. β-catenin is a key mediator of the Wnt signaling pathway and plays a crucial role in cell-cell adhesion, cell differentiation, and other cellular processes.
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Lab products found in correlation
Foxa2, by Abcam (1 mentions)
Mf52 n, by Mshot (1 mentions)
Hnf4a, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
αsma antibody, by Proteintech (1 mentions)
Bx53 biomicroscope, by Olympus (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Fitc conjugated goat anti mouse antibody, by Abcam (1 mentions)
γh2ax antibody, by Thermo Fisher Scientific (1 mentions)
Cy3 conjugated goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Cy3 conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Hdac2 antibody, by Proteintech (1 mentions)
Fluorescent mounting medium, by Solarbio (1 mentions)
Alexa fluor 488 conjugated antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated antibody, by Thermo Fisher Scientific (1 mentions)
Vimentin antibody, by Proteintech (1 mentions)
α tubulin antibody, by Cell Signaling Technology (1 mentions)
C myc antibody, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Zhongshan Golden Bridge Biotechnology (1 mentions)
γh2ax antibody, by Cell Signaling Technology (1 mentions)
7 protocols using β catenin antibody
1
Immunofluorescence Staining of Hepatic Markers
2
Immunofluorescence Staining for Tissue Analysis
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Immunofluorescence staining was previously described.30 Briefly, after rehydrated with gradient ethanol, sections were repaired with EDTA solution at 100°C for 15 minutes and then washed by PBS solution 3 times. Non‐specific binding sites were blocked by incubation with goat serum at room temperature for 30 minutes. After blocking, sections were incubated with the primary antibodies overnight at 4°C and with a secondary fluorescent antibody for 1 hour at 37°C, respectively. Then, sections were counter‐stained with DAPI (Beyotime Biotechnology) and evaluated by a fluorescent microscope (Olympus bx53 biomicroscope). The following primary antibodies were used: α‐SMA antibody (Proteintech), Collagen Type I (Collagen I) antibody (Proteintech) and β‐Catenin antibody (Proteintech).
3
Immunofluorescence Staining Protocol
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The slides were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 (Beyotime Biotechnology) for 30 min. After blocking in 1% BSA for 15 min at room temperature, cells were incubated with primary antibody overnight at 4 °C. The primary antibodies were as follows: γH2AX antibody (1:200 diluted, Thermo Fisher Scientific), USP43 antibody (1:50 diluted, Santa Cruz Biotechnology), HDAC2 antibody (1:100 diluted, Proteintech Group) and β-catenin antibody (1:50 diluted, Proteintech Group). Then cells were incubated with Cy3-conjugated goat anti-rabbit antibody (1:200 diluted, Invitrogen), Cy3-conjugated goat anti-mouse antibody (1:200 diluted, Invitrogen) or FITC-conjugated goat anti-mouse antibody (1:200 diluted, Abcam) for 60 min at room temperature in the dark. After washing with PBS, cells were counterstained with DAPI (Aladdin) to mark the nuclei. The slides were sealed with fluorescent mounting medium (Solarbio Science), and observed under the microscope.
4
Immunostaining Antibody Inventory for Germ Cell Research
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PPP6C antibody (rabbit, A300-844A; Bethyl Laboratories, Inc.); SYCP3 antibody (rabbit, NB300-231; Novus Biologicals); α-tubulin antibody (rabbit, 2144; Cell Signaling Technology, Inc.); β-actin antibody (mouse, 3700; Cell Signaling Technology, Inc.); Phospho-β-Catenin (Ser552) (D8E11) antibody (rabbit, 5651; Cell Signaling Technology, Inc.); SYCP3 antibody (mouse, sc-74569; Santa Cruz); γH2AX antibody (rabbit, 9718; Cell Signaling Technology, Inc.); MVH antibody (mouse, ab27591; abcam); SYCP1 antibody (rabbit, ab15090; abcam); SOX9 antibody (rabbit, AB5535, Sigma-Aldrich); PLZF antibody (goat, AF2944, R&D Systems); β-catenin antibody (rabbit, 51067-1-AP, Proteintech); ZO2 antibody (rabbit, 18900-1-AP, Proteintech); TEX14 antibody (rabbit, 18351-1-AP, Proteintech); Vimentin antibody (rabbit, 10366-1-AP, Proteintech); HA-Tag mab (mouse, AE008; ABclonal); c-Myc antibody (mouse, m4439; sigma); green-fluorescent Alexa Fluor® 488 conjugate of lectin PNA (L21409, Thermo). Horseradish peroxidase–conjugated secondary antibodies were purchased from Zhongshan Golden Bridge Biotechnology Co, LTD (Beijing). Alexa Fluor 488–conjugated antibody and Alexa Fluor 594–conjugated antibody were purchased from Life Technologies.
5
Immunohistochemical Analysis of GATA6, β-catenin, and ZEB1
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We deparaffinized Paraffin-embedded PC tissues continuous sections (4um) at 65°C for 2h and then washed them using ethanol and xylene. Antigen repair was done at high pressure for 2.5min for GATA6, β-catenin, and ZEB1. We then incubated sections with hydrogen peroxide (3%) and 10% normal goat serum for 15min and 20 min, respectively. The sections were also incubated with a GATA6 antibody (1:200) (CST, USA), β-catenin antibody (1:200) (Proteintech, USA) and ZEB1 (1:200) (Proteintech, USA) at 4°C overnight. We incubated secondary antibody using streptavidin–peroxidase reagent with sections for 15 min, and then added 3,3′-diaminobenzidine (DAB) for color reactions and visualized at ×20 magnification.
We conducted Immunohistochemistry (IHC) score as previously described.14 (link) GATA6, β-catenin and ZEB1 coloration ranges were used as the standard to assess the staining score defined as 1 (0–25%), 2 (25–50%), 3 (50–75%), 4 (75–100%)[0 (negative), 1 (weak), 2 (moderate) and 3 (strong)]. The two scores were the final score ranging between 0 to 7, and the final score≥ 3 was defined as positive expression.
We conducted Immunohistochemistry (IHC) score as previously described.14 (link) GATA6, β-catenin and ZEB1 coloration ranges were used as the standard to assess the staining score defined as 1 (0–25%), 2 (25–50%), 3 (50–75%), 4 (75–100%)[0 (negative), 1 (weak), 2 (moderate) and 3 (strong)]. The two scores were the final score ranging between 0 to 7, and the final score≥ 3 was defined as positive expression.
6
Adiponectin and TGF-β1 Modulate Vascular Remodeling
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Human adiponectin (Acrp30) and human TGF-β1 were purchased from Novoprotein (Beijing, China). Cell Counting Kit-8 (CCK-8) and EdU kits were procured from SolarBio (Beijing Solebo Technology Co., Ltd, Beijing, China). PCNA antibody, smooth muscle actin polyclonal (α-SMA) antibody, collagen type I (collagen I) rabbit polyclonal antibody, p38 MAPK antibody, p-p38 MAPK antibody, NF-κB antibody, p-NF-κB antibody, β-catenin antibody, p-β-catenin antibody, Notch1 antibody, and HRP-conjugated AffiniPure goat anti-mouse IgG (H + L) were obtained from Proteintech (Wuhan, China). SB203580 was purchased from MCE (Shanghai, China). Other chemicals used in this study, such as the BCA protein assay kit and SDS-PAGE gel separation kit, were acquired from ASPEN (ASPEN Biotechnology Co., Ltd, Wuhan, China).
7
Western Blot Protein Analysis
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Lysis buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.1% SDS, and 5 mmol/L EDTA (pH 8.0)] was prepared for the following experiment. After cells were treated by precooling phosphate buffer saline (PBS), but before transferred into the EP tube, protease inhibitors and RIPA lysis were added for the purpose of complete lysis. The supernatant after centrifugation (4°C, 15min, and 15000r/min) was stored at -80°C. Protein samples of 20 g was added to each hole and separated by 12% SDS-polyacrylamide gel electro-phoresis and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) which were subject to antibody reaction (internal reference = GAPDH). TLR4 antibody (Protein tech Wuhan, China), β-catenin antibody (Protein tech, Wuhan, China) and GAPDH antibody (Abcam, USA) (dilution concentration were 1:400, 1:500, 1:900, respectively) were used in this study. Membranes were blocked with 5% fat-free milk and then incubated with primary antibodies at 4°C overnight, followed by the incubation with secondary antibodies for two hours.
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