Cell culture media were removed from the devices, and samples in the microfluidic devices were first rinsed in 1X PBS by adding 70 µL of PBS into one port and another 50 µL into the opposite connected port of a media channel. Then, the cells were fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at room temperature. Next, 0.1% Triton-X 100 (Sigma-Aldrich, St. Louis, MO, USA) was added, and the device was incubated for 10 min before blocking by BSA 1% (Sigma-Aldrich cat no.: A5611) for two hours, followed by staining of cells for α-SMA (1:100, Abcam, Cat# ab197240.), Vimentin (1:200, Biolegend Cat# 677804. San Diego, CA, USA), YAP1 (1:200, Abcam, Cat# ab205270), CD44 (1:200, Abcam, Cat# ab194988), and CDH1 (1:200, Biolegend Cat# 324104). Nikon Ti2 confocal microscopes were used for imaging of the samples. The intensity of fluorescent signal, an indicator of protein expression, was analyzed on multiple z-stack. Finally, the images’ mean fluorescent intensity (MFI) was quantified using Cell-Sense software (Olympus, Japan).
Vimentin
Manufactured by BioLegend
Sourced in United States
Vimentin is a type III intermediate filament (IF) protein that is commonly used as a marker for mesenchymal cells. It is involved in maintaining the structural integrity of the cell and plays a role in cell migration and signaling.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by BioLegend (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Cellsense software, by Olympus (1 mentions)
α actinin, by Abcam (1 mentions)
Click it edu imaging kit, by Thermo Fisher Scientific (1 mentions)
Fluorescent conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Fluorescent conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Phospho myosin light chain 2 thr18 ser19, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by BioLegend (1 mentions)
Catalase, by Abcam (1 mentions)
Enhanced chemiluminescence substrate, by Bio-Rad (1 mentions)
Tnf α, by Abcam (1 mentions)
α sma, by BioLegend (1 mentions)
Fuji blue x ray film, by Bio-Rad (1 mentions)
Klotho, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
6 protocols using vimentin
1
Immunofluorescence Staining in Microfluidics
2
Histological Analysis of Myocardial I/R Injury
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Heart tissues obtained from I/R mice were embedded in paraffin after fixation in 4% paraformaldehyde. Heart sections (4 μm) were processed as previously described and stained with hematoxylin–eosin (HE), anti‐myeloperoxidase (MPO; Abbiotec), and inducible nitric oxide synthase (INOS) (eBioscience). Opositive cells and case areas were observed by a microscope. Heart tissue obtained from mice reperfused for 24 h was used for immunofluorescence colocalization to analyze IL‐38‐expressing cells after I/R. Antibodies used for immunofluorescence were as follows: CD3 (1:100; BioLegend), CD68 (1:100; Santa Cruz Biotechnology), α‐actinin (1:100; Abcam), and vimentin (1:100; BioLegend). The average number of positive cells in the infarct area was used for comparative analysis.
3
Immunofluorescence Staining of Cytoskeleton
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For immunofluorescence staining, fixed cultures were permeabilized with 0.25% Triton-X-100, blocked with 5% goat serum prior to incubation with primary antibodies. Primary antibodies for immunolabeling included: vimentin (AMF-17b, DSHB, Iowa City, IA, USA) Phospho-Myosin Light Chain 2 (Thr18/Ser19, Cell Signaling 3674, Danvers, MA, USA). The AMF-17b monoclonal antibody to vimentin was deposited by Fulton, A.B. Cultures were counterstained with DAPI (Biolegend, San Diego, CA, USA) to identify nuclei and fluorescent-conjugated Phalloidin (Invitrogen, Waltham, MA, USA) to label filamentous actin. Following primary antibodies, cultures were incubated with fluorescent conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA). To detect proliferating cells, cultures were EdU-labeled with 10 μM EdU for 30 min and the Click-iT EdU imaging kit (Invitrogen, Carlsbad, CA, USA) was used following manufacturer’s instructions. Images were taken on a confocal Zeiss 800. Z-stacks were collected at 0.33 μm and 3D-structural images were created from Zeiss 800 confocal microscope using Imaris ×64 v9.5.1 software surface rendering tool.
4
Quantitative Protein Expression Analysis
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The protein expression levels were determined by Western blotting. The samples were re-labeled for a blind analysis before the Western blot processing. Equal amounts of protein extracts were separated using sodium dodecyl sulfate (SDS)-PAGE and then electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with the primary antibodies for E-cadherin (#866702), vimentin (#677802), α-SMA (#904601) (Biolegend, San Diego, CA, USA), p53 (#sc-126), p16 (#sc-377412), p21 (#sc-6246), β-actin (#sc47778), GAPDH (#sc25778) (Santa Cruz Dallas, TX, USA), catalase (#ab16731), superoxide dismutase 1 (SOD1; #ab13498), Klotho (#ab203576), CD68 (#ab125212), TNF-α (#ab183218) (Abcam, Cambridge, MA, USA), Ly6G (#14-5931-82) (eBioscience, San Diego, CA, USA), Bax (#14796), Bcl-2 (#3498), cleaved Caspase 3 (#9664), and CHOP (#2895) (Cell Signaling Technology, Danvers, MA, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA). The blot signals were measured using enhanced chemiluminescence substrates (Bio-Rad) and developed onto a Fuji Blue X-ray Film. ImageJ software was used to quantify the protein expression bands.
5
Protein Expression Analysis by Western Blot
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Proteins in cells were resolved in lysis buffer which supplemented with 1 mM PMSF, and lysed for 15 min on ice. Briefly, collected the supernatant after centrifugation at 12,000×g for 10 min and diluted in 5×SDS-PAGE loading buffer (Biolegend, San Diego, CA), heated at 95 °C for 5 min and cooled on ice. Protein concentration of each sample was quantified by using of the BCA assay. Then these proteins were transferred onto the PVDF membrane (Bio-Rad, Hercules, CA) after separated. Then the membranes were blocked and reacted with first antibodies against CtBP2, E-cadherin, β-catenin, Vimentin (Biolegend, San Diego, CA) and β-actin (Abcam, Cambridge, MA) in a 4 °C refrigerator overnight. The β-actin was used for the purpose of an internal control. Following, the membranes were reacted with secondary HRP-conjugated antibody. The protein bands were visualized and imaged via chemiluminescence detection system (Tanon, Shanghai, China), and quantified by ImageQuant TL (GE healthcare life sciences, Pittsburgh, PA).
6
Multiplex Immunofluorescence Analysis of MPM
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For DBP and HTDBP, nuclei were stained with Hoechst 33342 (1:2000; Life Technologies) to determine total number of cells. A pan-cytokeratin antibody (#628608; 1:1000; Biolegend) was used to identify epithelioid MPM tumor cells parent population and vimentin (#677809; 1:1000; Biolegend) was used to identify sarcomatoid MPM parent population. Cytochrome c positive cells % was measured using cytochrome c-Alexa647 antibody (#612310; 1:2000; Biolegend). Western blotting was performed per manufacturer specifications (BIO-RAD). For Western blotting all antibodies were from Cell signaling Technology unless otherwise stated, β-Actin (#4967S 1:2000), AKT (#4685S 1:1000), phosphoSer473-AKT (#4685S 1:1000), BIM (#2933S 1:1000), BAK (#12105S 1;1000), BAX (#2772S 1;1000), BCL-xL (#2764S 1;1000), BCL-2 (#15071S 1;1000), cleaved Caspase 3 (#9661S 1:1000), MCL-1 (#39224S 1;1000; human specific for PDX samples), MCL-1 (#94296S 1;1000), PUMA (#4976S 1;1000), PARP (#9542S 1;1000), S6 (#2317S 1;1000) and phosphoSer235/236-S6 (#4857S 1;1000).
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