Protein β-catenin, CD14, p-ERK and p-Jun N-terminal kinase (p-JNK) expression in Wnt/β-catenin signaling pathways of SSCs after treatment for each group was detected by cell immunofluorescence. Briefly, a further three generations of SSCs were cultivated according to the grouping method. SSCs were fixed in precool methanol for 20 minutes. Sealed non-specific protein in 1% bovine serum albumin (BSA) for 1 hour. Cells were then incubated in diluting primary antibodies for 2 hours at 37 °C (rabbit anti-mouse β-catenin, CD14, p-ERK and p-JNK) (Abcam Inc., Cambridge, MA), followed by the secondary antibody for 1 hour at 37 °C [fluorescein labeled antibody to rabbit IgG (H + L); 1:100]. The cell nucleus was incubated with diamidino-phenyl-indole for 10 minutes at room temperature. Cells were washed 5 times by PBST between each step. Fluorescence was detected and recorded by the inverted fluorescence microscope.
Anti mouse β catenin
Manufactured by Abcam
Sourced in United States
Anti-mouse β-catenin is a primary antibody that specifically binds to the β-catenin protein in mouse samples. β-catenin is a key component of the Wnt signaling pathway and plays a role in cell-cell adhesion.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase hrp conjugated secondary antibody, by Beyotime (1 mentions)
Ecl solution, by Merck Group (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Alexa fluor 555 donkey anti rabbit igg, by Beyotime (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Beyotime (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Fluoromount, by Vector Laboratories (1 mentions)
Vectastain elite abc peroxidase kit, by Vector Laboratories (1 mentions)
Rabbit anti igf 1, by Abcam (1 mentions)
Vector sg, by Vector Laboratories (1 mentions)
Pecam 1, by Santa Cruz Biotechnology (1 mentions)
4 protocols using anti mouse β catenin
1
Investigating Wnt/β-Catenin Signaling in SSCs
2
Protein Expression Analysis of Xenografts
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Protein from xenografts was extracted using RIPA protein extract solution (Solarbio, Beijing, China). Protein concentration was determined by bicinchoninic acid protein assay kit (Thermo, USA). Samples were boiled with 4x loading buffer for 5 min. Aliquots of 15 μg of total protein were separated on 10% or 15% polyacrylamide gels. After electrophoresis, the samples were transferred to polyvinylidene difluoride (PVDF) membranes, blocked overnight at 4 °C in 5% nonfat milk/PBS. Membranes were then incubated for 2 h at room temperature with anti-mouse β-catenin (1:1000, Abcam, USA), anti-mouse survivin (1:1000, Abcam, USA), anti-mouse c-myc (1:1000, Abcam, USA), anti-mouse Cyclin D1 (1:800, Abcam, USA) or anti-mouse GAPDH (1:2000, CST, USA). Membranes were washed and incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibodies (Beyotime, Beijing, China), the proteins were visualised using an ECL solution (Millipore, USA) and imaged using a Tanon-5200 Multi system (Tanon, Shanghai, China).
3
Immunostaining of FABP7 and β-catenin
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Cells were seeded on the coverslips in 24-well plates and fixed with 4% paraformaldehyde. After 20 min, cells were permeabilized with phosphate-buffered saline (PBS) containing 0.2% Triton X-100 (PBS-T) for 5 min and then blocked with 1% bovine serum albumin in PBS-T for 10 min. Immunostaining was performed using primary antibodies, rabbit anti-FABP7 and mouse anti-β-catenin (Abcam, Cambridge, MA, USA), overnight at 4 °C. After PBS washing for 3 times, the slides were incubated with Alexa Fluor 555 donkey anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG (Beyotime, Beijing, China), respectively, at room temperature for 1 h. The slides were counterstained with DAPI (Beyotime, Beijing, China) and images were captured using the Zeiss microscope (Carl Zeiss, Jena, Germany).
4
Immunohistochemistry and Immunofluorescence Protocols
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For immunohistochemistry analysis, the sections were deparaffinized and boiled in a citrate buffer solution. The sections were stained using standard methods with rabbit anti-CD31 (PECAM-1, Santa Cruz Biotechnology, Inc. 1 : 100) as the primary antibody. The sections were incubated using a Vectastain Elite ABC-Peroxidase kit (Vector Laboratories, Burlingame, CA, USA), visualized using a Vector SG (Vector Laboratories), and counterstained with nuclear fast solutions (Vector Laboratories). For immunofluorescence, the sections were deparaffinized and boiled in a citrate buffer solution. They were stained using standard methods with rabbit anti-IGF-1 (dilution 1 : 100, Abcam, Cambridge, UK) and mouse anti-β-catenin (dilution 1 : 100, Abcam) as the primary antibodies. The sections were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse and Texas red-conjugated goat anti-rabbit secondary antibodies (dilution 1 : 100, Santa Cruz Biotechnology, Inc.). The sections were fixed with fluoromount (Vector Laboratories) and viewed under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany).
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