Tissues were perfused with PFA 4% for immunofluorescent labeling. Frozen OCT-embedded sections were cut to 5 μm and epitope retrieval was performed in citrate buffer (10 mM sodium citrate, pH 6.0). Sections were blocked with donkey antiserum 5% (Jackson ImmunoResearch Laboratories, code 017-000-121) and bovine serum albumin 1% % (Jackson ImmunoResearch Laboratories, code 001-000-162). Primary and secondary antibodies were dissolved in blocking buffer and incubated overnight at 4 °C. Labeling was performed with anti-actin antibody, diluted 1:500 (Abcam, ab20272), anti-caveolin-1 antibody-caveolae marker diluted 1:1000 (Abcam 18199); anti-caveolin-3, diluted 1:1000 (Abcam, ab2912) and with phalloidin-iFluor 405 reagent (Abcam, ab176752), as specified. Fluorescently labeled secondary antibodies (Jackson ImmunoResearch Laboratories) were dissolved in blocking buffer and applied for 2 h at room temperature. As indicated, the cell nuclei were counterstained with DAPI with blue fluorescence. Samples were mounted with Fluoromount mounting media (Thermo-Fisher, 00-4959-52). Images were acquired using a laser-scanning confocal microscope system (LSM 700, Carl Zeiss, Zeiss, Germany).
Ab2912
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab2912 is a primary antibody that recognizes the target protein. It is suitable for use in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ab11425, by Abcam (2 mentions)
Rabbit igg sc 2027, by Santa Cruz Biotechnology (2 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (2 mentions)
Quick start bradford dye reagent, by Bio-Rad (2 mentions)
Bis tris gel, by Bio-Rad (2 mentions)
Mouse igg sc 2025, by Santa Cruz Biotechnology (2 mentions)
Dynabeads protein g, by Santa Cruz Biotechnology (2 mentions)
Sc10646, by Santa Cruz Biotechnology (2 mentions)
Ac003, by Alomone (2 mentions)
Ac062, by Alomone (2 mentions)
Goat igg sc2028, by Santa Cruz Biotechnology (2 mentions)
Ma3 916, by Thermo Fisher Scientific (2 mentions)
A11070, by Thermo Fisher Scientific (2 mentions)
A 21237, by Thermo Fisher Scientific (2 mentions)
Image it fx signal enhancer, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Jackson ImmunoResearch (1 mentions)
Ab20272, by Abcam (1 mentions)
Lsm 5 pascal laser scanning confocal microscope, by Zeiss (1 mentions)
13 protocols using ab2912
1
Immunofluorescent Labeling of Cytoskeletal Proteins
2
Cardiac Protein Immunoprecipitation and Analysis
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The study was carried out following the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health and the Directive 2010/63/EU of the European Parliament. 6-8 weeks old male mice of strain C57BL6 were sacrificed by cervical dislocation and their hearts were harvested and snap frozen in liquid nitrogen and stored at -80 °C. Heart tissue was homogenized on a Precellys 24 and solubilized in ice-cold lysis buffer containing protease and phosphatase inhibitors. Tissue lysates were centrifuged to remove insoluble debris. For each tissue preparation produced, lysates derived from 5 mice were pooled and protein concentrations were measured by Quick Start Bradford Dye Reagent (Biorad). Solubilized heart tissue lysate was pre-cleared with Dynabeads protein G (Invitrogen) before incubation with primary antibody followed by binding to Dynabeads protein G, using either anti-KCNQ1 (10 μl SC10646, Santa Cruz), anti-CACNA1C (2 μl AC003, Alomone), anti-KCNH2 (2 μl AC062, Alomone), anti-CAV3 (2 μl ab2912, Abcam), anti-SNTA1 (2 μl ab11425, Abcam) or control IgG (1.5 μl goat IgG: SC2028, 1.5 μl rabbit IgG: SC2027, 1.5 μl mouse IgG: SC2025, Santa Cruz). After washing, bound proteins were eluted with 1× sample buffer containing 100 mM dithiothreitol (70 °C, 3 min) and separated by SDS-PAGE (4-15 % Bis-Tris gels, BioRad).
3
Papillary Muscle Contractility Assay
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Anterior and posteromedial papillary muscles were harvested from anaesthetized 11-week-old Wistar rats and mounted with sutures in a myobath system (Myobath II Multi-Channel Tissue Bath System, World Precision Instruments). Muscles were superfused with Tyrode's buffer solution and electrically stimulated at 0.5 Hz, while force was recorded during isometric contraction. Pre-load (diastolic wall stress) was set by stretching muscles. Diastolic, systolic, and developed force were sampled for 1 min every hour. Following 48 h of force recording, muscles were frozen and cut into transverse 20 µm sections, and t-tubules were labelled with Caveolin-3 antibody (Abcam, ab2912).
4
Immunohistochemical Analysis of Caveolin-3
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6-μm-thick cut sections were taken from the deparaffinized process described above and rehydrated positively charged slides for immunohistochemical assay. For antigen retrieval, the sections were kept in Antigen Unmasking Solution (Vector Laboratories) in 90 °C for 20 min and incubated an with anti-cav-3 antibody (1:500 dilution) (ab150077, Abcam, Cambridge, UK) for one night. It was incubated all sections with secondary antibody Alexa 488 (1:1000 dilution) (ab2912, Abcam, Cambridge, UK) and Cy3-conjugated donkey anti-mouse IgG antibody (1:1000 dilution) (ab150077, Abcam, Cambridge, UK) during one h. Finally, the sections were examined using a Zeiss LSM 5 PASCAL laser scanning confocal microscope.
5
Characterization of Exosome Origins
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Anti-CD81 (sc-166029), anti-CD9 (sc-13118), anti-Hsp70 (sc-32239), and anti-hnRNPA2/B1 (sc-53531) antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA). From Abcam (Cambridge, MA, USA) were anti-caveolin-1 (Cav-1, ab2910) and anti-caveolin-3 (Cav-3, ab2912). The antibodies used in flow cytometry to characterize the origin of exosomes were anti-human CD142-PE to monocytes and endothelial cells (#550313), anti-human CD146-PE-CF594 to endothelial cells (#564327), anti-human CD45-APC (#555485) to leucocytes and hematopoietic cells, and anti-human CD36-PerCP-Cy (#561536) to platelets from BD Biosciences (San Jose, CA, USA). Hsp70 was used as a specific marker of cardiomyocyte-derived exosomes [46 (link)]. The Megamix-Plus FSC Beads were acquired from BioCytex (BioCytex, Marseille, France) and aldehyde/sulfate latex beads with 4 μm (A37304) were from Invitrogen™ (Waltham, MA, USA), other reagents used are mentioned where required.
6
Histological Analysis of Ion Channels
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Masson's trichrome staining and double‐immunolabeling for ClC‐2 (goat polyclonal; SAB2501373, Sigma‐Aldrich), ClC‐3 (rabbit polyclonal; ACL‐001, Alomone Labs), SWELL1 (anti‐LRRC8A, rabbit polyclonal; AAC‐001, Alomone Labs), and Cav3 (mouse monoclonal; 610421, BD Biosciences; rabbit polyclonal; ab2912, Abcam) were performed on paraffin‐embedded sections of unstretched PV preparations. Images were collected using a Leica SP5 confocal microscope system under ×63 oil‐immersion objective and analyzed using the NIH ImageJ and Matlab software.
7
Immunoblot Analysis of Cellular Proteins
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Immunoblot was carried out as described in a previous report (Gao, 2016 b). In brief, following treatment with RIPA lysis buffer (P0013, Beyotime, China), total protein was quantitated with a BCA protein assay kit (P0012, Beyotime).
After separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the protein bands underwent transfer onto polyvinylidene fluoride (PVDF) membranes. This was followed by overnight incubation (4℃) with primary antibodies raised against integrin α1 (1:1,000; sc-271,034, Santa Cruz, USA), caveolin-1 (1:1,000; sc-894, Santa Cruz), caveolin-2 (1:1,000; ab2911; Abcam, UK), caveolin-3 (1:1,000; ab2912; Abcam), and GAPDH (1:5,000; AP0063; Bioworld Technology, USA). Following washing with PBS, goat anti-mouse (GAM007; MultiSciences Technology, USA) or anti-rabbit (GAR0072; MultiSciences Technology) IgG-HRP was added for 1 h at ambient. Finally, Immobilon™ Western Chemiluminescent HRP substrate reagent (EMD Millipore, USA) was employed for development. Immunoreactive bands were captured and assessed on a Bio-Rad Gel Doc Imaging System (Bio-Rad Laboratories, USA).
After separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the protein bands underwent transfer onto polyvinylidene fluoride (PVDF) membranes. This was followed by overnight incubation (4℃) with primary antibodies raised against integrin α1 (1:1,000; sc-271,034, Santa Cruz, USA), caveolin-1 (1:1,000; sc-894, Santa Cruz), caveolin-2 (1:1,000; ab2911; Abcam, UK), caveolin-3 (1:1,000; ab2912; Abcam), and GAPDH (1:5,000; AP0063; Bioworld Technology, USA). Following washing with PBS, goat anti-mouse (GAM007; MultiSciences Technology, USA) or anti-rabbit (GAR0072; MultiSciences Technology) IgG-HRP was added for 1 h at ambient. Finally, Immobilon™ Western Chemiluminescent HRP substrate reagent (EMD Millipore, USA) was employed for development. Immunoreactive bands were captured and assessed on a Bio-Rad Gel Doc Imaging System (Bio-Rad Laboratories, USA).
8
Multicolor Protein Localization Imaging
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Cell samples underwent treatment with 5 µg/ml of cholera toxin B-fluorescein isothiocyanate (CTB-FITC; c34775; Thermo Fisher Scientific, USA) at room temperature for 30 min. Then, cells were permeabilized using 0.1% Triton X-100 after fixation with 4% paraformaldehyde. This was followed by successive incubations with primary (4 °C, overnight) and secondary (4 °C, 2 h in the dark) antibodies. The primary antibodies were raised against β-Tubulin (1:500; ab009, MultiSciences Technology), F-actin (1:500; ab205; Abcam) and caveolin-3 (1:1,000; ab2912; Abcam). The secondary antibodies were as follows: Donkey anti-rabbit IgG-Alexa Fluor® 647 (A31573; Thermo Fisher Scientific) and Goat anti-mouse IgG-Alexa Fluor® 555 (A21424; Thermo Fisher Scientific). Finally, DAPI (D9542; Sigma-Aldrich) was used for counterstaining at ambient for 20 min before observation under an A1 laser confocal microscope (Nikon, Japan).
9
Cardiac Protein Immunoprecipitation and Analysis
10
Western Blot Analysis of COX-2, Cav-3 in Spinal Cord
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Spinal cords were harvested, homogenized, and lysed with protease inhibitor-containing RIPA buffer. The concentrations of protein lysate were calculated using the Pierce BCA Protein Assay Kit (Thermo Scientific, USA). A total of 20 μg ~ 30 μg of proteins was separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with the following primary antibodies: anti-COX-2 (ab52237, abcam, USA), anti-Cav-3 (ab2912, abcam, USA) and anti-GAPDH (G9545, Sigma, USA). After overnight incubation, the membranes were blotted with HRP- conjugated secondary antibody (Jackson ImmunoResearch, USA), and visualized using Clarity ECL Westerm Blotting Substrate (Bio-rad, USA).
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