Antibodies used in this study include mouse monoclonal anti-CK8 (1/200 dilution) (Covance, CA), rabbit polyclonal anti-Vim (1/50 dilution) (Santa Cruz Biotechnology, CA), Alexa 488 goat anti-rabbit, and Alexa 568 goat anti-mouse (Invitrogen, CA). All secondary Alexa Fluor antibodies were used at 1/200 dilution. Fluoro-gel II with DAPI (Electron Microscopy Sciences, PA) was used for mounting.
Anti vim
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-VIM is a laboratory antibody product used for the detection of Vimentin (VIM) protein in biological samples. VIM is a type III intermediate filament protein involved in maintaining cell structure and integrity. The Anti-VIM product can be utilized in various research and diagnostic applications that require the identification and quantification of VIM expression.
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Lab products found in correlation
Anti akt, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti gata4, by Santa Cruz Biotechnology (2 mentions)
Sc 6260, by Santa Cruz Biotechnology (2 mentions)
Goat anti mouse alexa 568, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit alexa 488, by Thermo Fisher Scientific (1 mentions)
Fluoro gel 2 with dapi, by Electron Microscopy Sciences (1 mentions)
Mouse monoclonal anti ck8, by Fortrea (1 mentions)
Smad4, by Proteintech (1 mentions)
Anti e cad, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Anti ck19, by Santa Cruz Biotechnology (1 mentions)
Anti mmp2, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti phosphorylated smad2, by Cell Signaling Technology (1 mentions)
Sb431542, by Merck Group (1 mentions)
Anti ppm1a, by Cell Signaling Technology (1 mentions)
Anti phosphorylated smad3, by Cell Signaling Technology (1 mentions)
Anti phosphorylated smad2 3, by Santa Cruz Biotechnology (1 mentions)
Sd 208, by Merck Group (1 mentions)
10 protocols using anti vim
1
Immunofluorescence Staining of Cytoskeletal Proteins
2
Smad4 Variant Expression and E-cadherin/Vimentin Analysis
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The A549 cells stably expressed wildtype Smad4 and its variants, which were used for the Western blot experiments. Collect and lyse cells with RIPA buffer for 30 min on ice. Total protein lysis was used to measure target protein expression. The anti-E-cad (Santa Cruz, Cat#sc-8426) and anti-VIM (Santa Cruz, Cat#sc-373717) antibodies were purchased from Santa Cruz. The anti-GAPDH (Cat#10494-1-AP) and Smad4 (Cat#10231-1-AP) antibodies were purchased from Proteintech. Load 30 μg total protein into 10% SDS-PAGE gel and run for 90 min at 120 V. Transfer the gel on the PVDF membrane and then incubate with 5% nonfat milk for 30 min by slowly shaking at room temperature. We then incubated the primary antibodies at 4°C in a freezer overnight and washed with TBST for 10 min, repeating 3 times. The secondary antibodies were then incubated at room temperature for 1 h by slowly shaking. The images of membrane were taken with a standard chemiluminescence.
3
Inhibition of TGF-β Signaling Pathway
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Human recombinant TGF-β1 was obtained from R&D Systems (Minneapolis, MN). The TGF-β RI (TβRI) kinase inhibitor SB431542 was obtained from Millipore (Billerica, MA), and SD-208 was purchased from Sigma (St Louis, MO). The following antibodies were used in this study: anti-PPM1A, anti-MMP2, anti-MMP9, anti-Smad2, anti-Smad3, anti-phosphorylated Smad2 and anti-phosphorylated Smad3 (Cell Signaling Technology, Beverly, MA) as well as anti-E-cadherin, anti-GAPDH, anti-CK19, anti-phosphorylated Smad2/3 and anti-Vim (Santa Cruz, CA, USA). SB431542 was applied during the TGF-β1 perfusion.
4
Western Blot Analysis of Signaling Proteins
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Cells were seeded at equal densities in complete media and the media exchanged 24 h later for EGF-free media +/− inhibitor for an additional 24 h growth. Cells were scraped into 1X PBS and lysed in RIPA buffer containing 1 mM PMSF (Sigma), 1 mM Na3VO4 (Sigma) and 1x cOmplete Protease Inhibitor cocktail (PI) (Roche, Indianapolis, IN, USA). Twenty milligrams protein was resolved by SDS-PAGE using NuPAGE 4–12% Bis-Tris protein gels (Life Technologies), transferred onto PVDF (Millipore, Billerica, MA, USA) and blocked in 5% BSA. Following overnight blotting with primary antibody, protein was detected by ECL using horseradish peroxidase-conjugated secondary antibodies (Sigma). Primary antibodies used in this study are: anti-RAS (#3965), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370S), anti-p44/42 MAPK (ERK1/2) (#4696), anti-PI3K p110α (#4255), anti-phospho-AKT (Ser473) (#4051), anti-AKT (#4685), anti-C-MYC (#9402), anti-NDRG1 (#5196), anti-GAPDH (#2118) (Cell Signaling Technologies, Danvers, MA, USA), anti-BRD9 (#A303-781A, Bethyl Laboratories, Montgomery, TX, USA), anti-BRG1 (#sc-17796) and anti-VIM (#sc-6260) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
5
Comprehensive Protein Expression Analysis
6
Immunofluorescence Staining Protocol for Protein Detection
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Immunofluorescence staining was performed using a similar protocol to the one described previously (Chau et al., 2014 (link)). The primary antibodies used in this study were anti-WT1 (Abcam, ab89901, 1:1000), anti-Ki67 (Abcam, ab15580, 1:1000), anti-GFP (Abcam, ab5450, 1:1000), anti-PAX2 (Biolegend, 901001, 1:100), anti-MF20 (DSHB, Ab_2147781, 1:20), anti-VIM (Santa Cruz Biotechnology, sc-7557, 1:100), anti-CDH1 (BD, 610181, 1:100), anti-RALDH2 (Santa Cruz Biotechnology, sc-22591, 1:200), anti-GATA4 (Santa Cruz Biotechnology, sc-25310, 1:100), anti-TCF4 (Cell Signaling Technology, C48H11, 1:100) and anti-MYOD (Santa Cruz Biotechnology, sc-32758, 1:100). Alexa-Fluor 488- or 594-conjugated antibodies were used as secondary antibodies. Sections were stained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted using VectorShield. A Zeiss Axioplan II microscope was used to view immunofluorescence and H&E-stained sections. Image capture was performed using the open source microscopy software: uManager.
7
Multicellular Protein Expression Profiling
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Cells were incubated with anti-panCK (1:200, sc-8018; Santa Cruz, CA), anti-VIM (1:500, sc-6260; Santa Cruz), anti-ASMA (1:200, A5228; Sigma, MA), and anti-FAP (1:100, ab53066; Abcam, Cambridge, UK) at RT for 3 h. The anti-mouse IgG-Cy3 (1:2,000, 115-166-071; Jackson ImmunoResearch Laboratories, PA) or anti-rabbit IgG-FITC (1:2,000, ab6717; Abcam) was applied at RT for 1 h. The anti-CD10-FITC (1:5, 21270103; Invitrogen, Thermo Fisher Scientific, MA) and anti-GPR77 (1:30, 342402; Biolegend, CA) were incubated with cells at 4°C overnight. The nuclei were stained with Hoechst dye (1:1,000; Invitrogen). The fluorescence signals were captured with a confocal microscope (LSM800, Carl Zeiss Microscopy, Jena, Germany).
8
Antibody Panel for Cell Characterization
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9
Western Blot Analysis of Endothelial Cell Markers
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Isolated ECs of mouse aortae, mouse aortae (whole, TA and AA), human ECs (HUVECs and HAECs) and human renal arteries were homogenized in ice-cold 1X RIPA lysis buffer supplemented with Complete Protease Inhibitor cocktail (Sigma-Aldrich) and phosSTOP phosphatase inhibitor (Roche, Basel, Switzerland). Protein concentrations were determined by a BCA protein assay kit (Pierce Biotechnology, Rockford, USA). Equal amount of proteins were loaded and resolved by a 10% SDS-polyacrylamide gel and transferred onto an Immobilon-P polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA, USA). Nonspecific binding sites were blocked by 3% BSA in TBS containing 0.05% Tween-20. The blots were incubated overnight at 4 °C with primary antibodies: anti-SOX4 (1:200; sc-130633; Santa Cruz Biotechnology, Texas, USA), anti-eNOS (1:1000; 610297; BD Transduction Laboratory, San Diego, USA), anti-PECAM-1 (1:500; sc-1506-R; Santa Cruz Biotechnology), anti-VIM (1:500; sc-6260; Santa Cruz Biotechnology) and anti-GAPDH (1:1000; 2118S; Cell Signaling Technology, Massachusetts, USA). Bound antibodies were incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) at room temperature for 1 h. Protein bands were later visualized by enhanced chemiluminescence (Cell Signaling Technology) with the aid of ChemiDoc™ Imaging System (Bio-Rad).
10
Immunocytochemical Analysis of Human Sertoli and Germ Cells
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For immunocytochemical staining, the freshly isolated and cultured human Sertoli cells as well as human male germ cells were fixed with 4% paraformaldehyde (PFA) for 30 min, washed three times with cold phosphate-buffered saline (PBS) and permeabilized in 0.4% triton-X 100 (Sigma) for 5 min. After washing with PBS, the cells were blocked in 2% BSA for 30 min and followed by incubation with primary antibodies, including anti-GATA4 (Santa Cruz), WT1 (Santa Cruz), GDNF (Santa Cruz), anti-SCF (Sigma), anti-BMP4 (Abcam), anti- VIM (Santa Cruz), anti-PCNA (Sigma), anti-SMA (smooth muscle alpha actin, Abcam) and CYP11A1 (cholesterol side-chain cleavage enzyme, Abcam) at a dilution with 1:200 overnight at 4°C. Isotype IgGs for the first antibody were used as the negative controls. After extensive washes with PBS for 30 min, the cells were incubated with the secondary antibody IgG (Sigma) conjugated with fluorescein isothiocyanate (FITC) or rhodamine at a 1:200 dilution for 1 hour at room temperature. DAPI (4,6-diamidino-2-phenylindole) was used to label the nuclei, and the cells were observed for epifluorescence under a fluorescence microscope (Leica).
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