We seeded EC‐1 cells in 24‐well culture dishes and treated with TGF‐β1 as mentioned above. After the treatment, the medium was discarded, and the EC‐1 cells were washed three times with PBS. Cells were stereotyped in PBS with 4% paraformaldehyde for 20 minutes and permeabilized in 0.5% Triton X‐100/PBS for 5 minutes at 23‐28°C. We blocked the cells with 2% bovine serum albumin in PBS for 30 minutes and then incubate the cells with anti‐E‐cadherin (1:100; Proteintech) and anti‐vimentin (1:100; Boster, Wuhan, China) antibodies at 37°C for 2 h. Finally, after washing with PBS, the cells were stained with a combination of the secondary antibodies conjugated to fluorescein isothiocyanate and tetramethyllrhodamine for 1 hour. Nuclei were labeled with 4',6‐diamidino‐2‐phenylindole and observed with the microscope (Olympus Corporation, Tokyo, Japan) at a multiple of ×200.
Anti vimentin
Manufactured by Boster Bio
Sourced in China
Anti-Vimentin is a primary antibody used in various laboratory techniques, including immunohistochemistry and Western blotting. It specifically binds to the vimentin protein, which is a type III intermediate filament found in various cell types. Anti-Vimentin can be used to detect and analyze the expression of vimentin in biological samples.
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Lab products found in correlation
Anti keratin, by Boster Bio (2 mentions)
Alexa fluor 488 anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Anti e cadherin, by Proteintech (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
D luciferin, by GoldBio (1 mentions)
Anti sqstm1 p62, by Cell Signaling Technology (1 mentions)
Dharmafect, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Merck Group (1 mentions)
H2dcfda, by Merck Group (1 mentions)
Anti lc3, by Cell Signaling Technology (1 mentions)
Pyronin y, by Merck Group (1 mentions)
Anti atg5, by Cell Signaling Technology (1 mentions)
Polybrene, by Yeasen (1 mentions)
Bafilomycin a1, by Sangon (1 mentions)
Pepstatin a, by Sangon (1 mentions)
Anti beclin 3495, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti mouse, by Boster Bio (1 mentions)
Propidium iodide, by Yeasen (1 mentions)
Buthionine sulfoximine, by Merck Group (1 mentions)
8 protocols using anti vimentin
1
Immunofluorescence Assay for Epithelial-Mesenchymal Transition
2
Comprehensive Cell Autophagy Assay
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H2DCFDA, rapamycin, pyronin Y, and buthionine sulfoximine were obtained from Sigma-Aldrich Co. (USA). Bafilomycin A1 and pepstatin A were obtained from Sangon Biotech Co. (China). D-Luciferin was obtained from Goldbio Co. (USA). Polybrene, Hoechst 33342, and propidium iodide (PI) were obtained from Yeasen Biotech Co. (China). Transfection reagents DharmaFECT, Lipofectamine 2000, and Alexa Fluor 488 and 567 secondary antibodies were obtained from Thermo Fisher Scientific Inc. (USA, 1:500). Anti-LC3 (#3868, 1:1000), anti-Beclin (#3495, 1:1000), anti-ATG5 (#12994, 1:1000), anti-Oct-4 (#2750, 1:1000), and anti-p62/SQSTM1 (#7695, 1:1000) antibodies were obtained from Cell Signaling Technology (USA). Anti-RB1CC1 (sc-22709, 1:100), anti-Ki-67 (sc-23900, 1:100), and HRP-conjugated goat anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology (USA). Anti-vimentin, HRP-conjugated goat anti-mouse, and rabbit anti-goat secondary antibodies were obtained from Boster Co. (China). Anti-β-tubulin (1:1000) and anti-β-actin (1:1000) antibodies were obtained from Transgene Biotech Co. (China). Anti-NRF2 (WL02135, 1:2000), anti-NOTCH1 (WL01991, 1:500), and anti-Lamin B (WL01775, 1:500) antibodies were obtained from Wanleibio Co. (China).
3
Comprehensive Protein Expression Analysis
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Following antibodies were used in western blot: anti-CCDC50 (ab127169, Abcam), anti-ZNF395 (11759–1-AP, Proteintech), anti-HnRNP A1 (ab5832, Abcam), anti-β-actin (BM0627, Boster), anti-PCNA (10205–2-AP, Proteintech), anti-Cyclin D1 (60186–1-Ig, Proteintech), N-Cadherin (22018–1-AP, Proteintech), anti-Vimentin (BM0135, Boster), anti-ZEB1 (3396, Cell Signaling Technology), anti-VEGF (19003–1-AP, Proteintech), goat anti-mouse IgG HRP-linked whole antibody (31,430, Thermo Scientific), and goat anti-rabbit secondary antibody (31,460, Thermo Scientific).
4
Western Blot Analysis of Cellular Markers
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Western blot analysis was performed essentially according to an established procedure [18 (link)]. The primary antibodies used were as follows: anti-β-actin (Sigma, St. Louis, MO), anti-α-SMA (Sigma, St. Louis, MO), anti-E-cadherin (BD Transduction Laboratories, Lexington, KY), anti-fibronectin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-HFE (Santa Cruz Biotechnology, Santa Cruz, CA), anti-vimentin (Boster, Wuhan, China), and anti-HIF-1α (Millipore, USA). Quantifications were performed by measuring band intensities using Image J analysis software.
5
Protein Extraction and Western Blot Analysis
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Cells were harvested and lysed in radio immunoprecipitation assay (RIPA) buffer (Solarbio, China) supplemented with phenylmethylsulfonyl fluoride (PMSF) (a serine protease inhibitor, Solarbio, China) for 30 min. BCA kit (Solarbio, China) was used to determine the concentration of protein in the supernatant. Proteins were separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane (Merck Millipore, USA), and then incubated overnight with primary antibody (4 °C) after being blocked for 2 h in nonfat milk solution (5%). The membrane was reacted with TBST-diluted HRP-conjugated secondary antibody for 2 h at room temperature. Protein bands were detected by Amersham Imager 600 System (General Electric Company, USA) using ECL Substrate (Beyotime Biotech, China), and quantified with Image J software. The primary antibodies contain anti-GAPDH (BBI, China), anti-GLUD1 (BBI, China), anti-E-cadherin (1:1000, Abcam, USA), anti-Vimentin (1:1000, Boster, China), anti-Cytochrome C (ProteinTech, USA), anti-BCL2 (ProteinTech, USA), anti-BAX (Wanleibio, China), anti-Caspase 3 (Wanleibio, China), anti-JNK (Wanleibio, China), anti-phospho-JNK (Wanleibio, China), anti-p53 (Cell Signaling Technology, USA), anti-p38 (Cell Signaling Technology, USA) and anti-phospho-p38 (Cell Signaling Technology, USA).
6
Immunofluorescence Staining of HDPFs
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HDPFs at the third passage were fixed with 4 % paraformaldehyde (PFA) for 30 min at room temperature and then incubated in PBS containing 0.4 % Triton X-100 for 10 min on ice and then blocked with 2 % bovine serum albumin (BSA) for 60 min at 37 °C.
After the blocking step, the cells were incubated with primary antibody-anti-vimentin (1:100, Boster, Wuhan, China), anti-keratin (1:100, Boster) at 4 °C overnight; PBS was used as the negative control. The cells were then washed with PBS and incubated for 1 h with the secondary antibodies, namely anti-mouse IgG Alexa Fluor-488 or at antirabbit IgG Alexa Fluor-594 1:1000 at room temperature (Life Technologies, Paisley, UK). Glass cover slips were mounted using mounting media supplemented with DAPI stain (VectorLabs, Peterborough, UK) and preparations imaged under a fluorescent microscope (Olympus, Tokyo, Japan).
After the blocking step, the cells were incubated with primary antibody-anti-vimentin (1:100, Boster, Wuhan, China), anti-keratin (1:100, Boster) at 4 °C overnight; PBS was used as the negative control. The cells were then washed with PBS and incubated for 1 h with the secondary antibodies, namely anti-mouse IgG Alexa Fluor-488 or at antirabbit IgG Alexa Fluor-594 1:1000 at room temperature (Life Technologies, Paisley, UK). Glass cover slips were mounted using mounting media supplemented with DAPI stain (VectorLabs, Peterborough, UK) and preparations imaged under a fluorescent microscope (Olympus, Tokyo, Japan).
7
Immunofluorescence Assay for Podocyte Characterization
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Podocytes in six-well plates were xed with 4% paraformaldehyde at room temperature for 30 min, incubated in phosphate-buffered saline (PBS) containing 0.4% Triton X-100 for 10 min, and blocked with 2% bovine serum albumin at 37°C for 60 min. The cells were then incubated with the primary antibodies anti-vimentin (1:100; Boster Biological Technology, Wuhan, China) and anti-keratin (1:100; Boster Biological Technology) at 4°C overnight. PBS was used as the negative control. Then, the cells were washed with PBS and incubated with anti-mouse IgG Alexa Fluor 488 or anti-rabbit IgG Alexa Fluor 594 (1:1000; Life Technologies, Paisley, UK) for 1 h at room temperature. The glass cover slides were installed using the installation medium with 4 ,6-diamidino-2-phenylindole dye (Vector Labs, Peterborough, UK) and prepared for imaging under a uorescence microscope under ⊆200 magni cation (Olympus, Tokyo, Japan).
8
Protein Extraction and Western Blotting
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Total protein was extracted in radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentrations were quantified by bicinchoninic acid (BCA) assay (Beyotime Institute of Biotechnology). Western blotting was performed as previously described. 22 The following primary antibodies were used: anti-MMP-2 (1:500; Bioworld Technology, Atlanta, GA, USA), anti-MMP-9 (1:500; Bioworld Technology), anti-E-cadherin (1:300; Boster, Wuhan, China), anti-Vimentin (1:200; Boster), anti-phosphorylated AKT (1:500; Cell Signaling Technology, Beverly, MA, USA), anti-AKT (1:1000; Cell Signaling Technology) and antiβ-actin (1:300; Zhongshan Golden Bridge, Beijing, China). Protein bands were quantified by densitometry using Quantity One 4.6.2 (Bio-Rad Laboratories, Hercules, CA, USA) and normalized to β-actin or total AKT.
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