DMSO, G418, and doxycycline hyclate (DOX) were purchased from Nacalai Tesque (Kyoto, Japan), Adipogen Life Sciences (Liestal, Switzerland), and Cayman Chemical Company (Ann Arbor, MI, USA), respectively. The following antibodies were used: anti‐FLAG (Sigma‐Aldrich, St. Louis, MO, USA), anti‐HA (Roche Applied Science, Penzberg, Germany), anti‐Myc (Santa Cruz Biotechnology, Inc, Dallas, TX, USA), anti‐Arp5 (Proteintech, Rosemont, IL, USA), anti‐GAPDH (Thermo Fisher Scientific, Waltham, MA, USA), anti‐ACTA2 (Sigma‐Aldrich), anti‐Collagen I (Novus Biologicals, Centennial, CO, USA), and anti‐GFP (Proteintech) antibodies.
Anti acta2
Manufactured by Merck Group
Sourced in United States
Anti-ACTA2 is a laboratory reagent used in research applications. It is a monoclonal antibody that specifically binds to the ACTA2 protein, which is involved in cellular processes. Anti-ACTA2 can be used to detect and quantify ACTA2 expression in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west femto substrate, by Thermo Fisher Scientific (2 mentions)
Anti s6 ribosomal protein, by Cell Signaling Technology (2 mentions)
Antihuman cleaved caspase 3, by Abcam (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Anti p70s6k, by Cell Signaling Technology (2 mentions)
Anti phospho p70s6k, by Cell Signaling Technology (2 mentions)
Anti gpnmb, by Cell Signaling Technology (2 mentions)
Anti phospho s6, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Nacalai Tesque (1 mentions)
Anti gfp, by Proteintech (1 mentions)
Anti collagen 1, by Novus Biologicals (1 mentions)
Anti ha, by Roche (1 mentions)
Anti arp5, by Proteintech (1 mentions)
Anti myc, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Anti mmp2, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
6 protocols using anti acta2
1
Cell Line Characterization Using Antibodies
2
Western Blot Analysis of Protein Expression
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Protein concentrations in lysates were quantified, and proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‒PAGE). After separation, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Inc. and incubated with a primary antibody followed by a secondary antibody. Luminescence was visualized with Enhanced Chemiluminescence Substrate (ECL) (Amersham Biosciences, Sweden). The results of western blotting were analyzed with NIH ImageJ 1.62 software. The following antibodies were used: anti-ACTG1 (actin gamma 1) and anti-ACTA2 (alpha-smooth muscle actin) from Sigma–Aldrich, anti-AZIN1 from Abbexa, and anti-ADAR1, anti-Myosin-9, anti-GAPDH, anti-α-Tubulin, anti-snail, anti-slug, anti-MMP2, anti-MMP9 and anti-Rpb1 CTD from Cell Signaling.
3
Protein Expression Analysis in Organoids
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In vitro and grafted organoids were lysed in RIPA buffer containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo FisherScientific), using a glass Dounce homogenizer (DWK Life Sciences Kimble™) on ice. Following protein quantification, SDS-PAGE gel run and western blotting were performed. The following antibodies from Cell Signaling Technology were used: antiribosomal protein S6 (#2217, clone 5G10, 1:1000), antiphospho S6 (#2211, 1:1000), antiphospho P70S6K (#9205, 1:1000), anti-P70S6K (#9202, 1:2000), anti-β Actin (#4967, 1:1000), anti-GPNMB (#38313, clone E4D7P, 1:700). Anti-ACTA2 was from Sigma-Aldrich (#F3777, clone 1A4, 1:2000), antihuman pro-Caspase 3 was purchased from ThermoFisher Scientific (#MA1-41163, clone 31A893, 1:400), antihuman cleaved Caspase 3 was purchased from Abcam (ab2302, 1:200), antihuman p21CIP1 was purchased from Novus Biologicals (#AF1047, 1:400). Primary antibodies were detected with peroxidase-conjugated antirabbit or anti-mouse IgG (1:3000) and visualized with SuperSignal West Femto Substrate (ThermoFisher Scientific #34094). Blots were quantified using ImageJ v1.51 W (https://imagej.nih.gov/ij/ ).
4
Comprehensive Immunohistochemistry Protocol
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Cryosections were stained with the following primary antibodies: anti-ACTA2 (1:500; Sigma-Aldrich, F3777 FITC-conjugated or C6198 Cy3-conjugated), anti-vimentin (1:500; Cell Signaling, 5741), anti-calponin (1:500; Abcam, ab46794), anti-collagen type I alpha 2 (1:500; Rockland, 600-401-103-0.1), anti-β-GAL (1:1000; Thermo Fisher Scientific, PA1-21477), anti-EpCAM (1:200; eBioscience, 14-5791-82), anti-GFP/YFP (1:1000; Nacalai Tesque, 0440484), anti-CK5 (1:2000; Covance, PRB-160P), and anti-CK8 (1:500; Developmental Studies Hybridoma Bank, TROMA-1). Secondary antibodies for double labeling were donkey anti-rabbit IgG (H+L) Alexa Fluor 555 (Invitrogen, A-31572), Alexa Fluor 488 (Invitrogen, A-21206) or Alexa Fluor 647 (Invitrogen, A-31573), and goat anti-rat IgG (H+L) Alexa Fluor 555 (Invitrogen, A-21434), all used at 1:1000. Nuclei were counterstained with DAPI.
5
Protein Expression Analysis in Organoids
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In vitro and grafted organoids were lysed in RIPA buffer containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo FisherScientific), using a glass Dounce homogenizer (DWK Life Sciences Kimble™) on ice. Following protein quantification, SDS-PAGE gel run and western blotting were performed as described (25) . The following antibodies Cell Signaling Technology were used: anti-ribosomal protein S6 (#2217), anti-phospho S6 (#2211), anti-phospho P70S6K (#9205), anti-P70S6K (#9202), anti-β Actin (#4967), anti-GPNMB (#38313). Anti-ACTA2 was from Sigma-Aldrich (#F3777), anti-human cleaved Caspase 3 was purchased from Abcam (ab2302). Primary antibodies were detected with peroxidase-conjugated anti-rabbit or anti-mouse IgG (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
(1:3000) and visualized with SuperSignal West Femto Substrate (ThermoFisher Scientific #34094). Blots were quantifi ed using ImageJ (https://imagej.nih.gov/ij/).
(1:3000) and visualized with SuperSignal West Femto Substrate (ThermoFisher Scientific #34094). Blots were quantifi ed using ImageJ (https://imagej.nih.gov/ij/).
6
Intracellular Staining of Organoids
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In each experiment, five organoids of each genotype were incubated with TrypLE Select (Thermo Fisher Scientific #12563011) in a shaker at 37°C for 10 min. The digestions were passed through a 40-µm cell strainer (Millipore Sigma #CLS431750), and washed (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted October 20, 2021. ; https://doi.org/10.1101/2021.10.20.465120 doi: bioRxiv preprint with PBS. Resulting single cell suspensions were collected by centrifugation at 400g for 5 min. Intracellular stainings with anti-PMEL antibody HMB-45-FITC (Novus Biologicals #NBP2-34638F) and with anti-ACTA2 (Sigma-Aldrich #C6198) were performed using the PerFix-nc Kit (Beckman Coulter #B31167), for 30 min at 4 °C in supplemented PBS containing 2 mM EDTA and 2% FBS. Analysis was performed on LSRII (Becton Dickinson) equipped with three lasers. Data were collected using FacsDIVA software. Sorting gates were defined on the basis of fluorescence-minus-one control stains.
The copyright holder for this preprint this version posted October 20, 2021. ; https://doi.org/10.1101/2021.10.20.465120 doi: bioRxiv preprint with PBS. Resulting single cell suspensions were collected by centrifugation at 400g for 5 min. Intracellular stainings with anti-PMEL antibody HMB-45-FITC (Novus Biologicals #NBP2-34638F) and with anti-ACTA2 (Sigma-Aldrich #C6198) were performed using the PerFix-nc Kit (Beckman Coulter #B31167), for 30 min at 4 °C in supplemented PBS containing 2 mM EDTA and 2% FBS. Analysis was performed on LSRII (Becton Dickinson) equipped with three lasers. Data were collected using FacsDIVA software. Sorting gates were defined on the basis of fluorescence-minus-one control stains.
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