Mouse embryonic fibroblasts cultured on glass coverslips were permeabilized in 1% Triton X-100 in phosphate-buffered saline (PBS), washed in PBS, blocked in 2% fetal bovine serum (FBS)–PBS, incubated with rabbit anti-Cx43 (Invitrogen, Carlsbad, CA), diluted 1:200 in PBS at room temperature for 2 h, washed in PBS, incubated in Alexa Fluor goat anti-rabbit immunoglobulin G (Molecular Probes, Eugene, OR), washed in PBS, and then stained with 1 mg/ml Hoechst 33258 diluted 1:1000 in PBS for 5 min at room temperature. Coverslips were mounted and stored at 4°C overnight. Cells were imaged on a Leitz Dialux 20 microscope with fluorescence and camera attachments (Leitz, Rockleigh, NJ).
Rabbit anti cx43
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Rabbit anti-Cx43 is a primary antibody that specifically binds to the Connexin 43 (Cx43) protein. Connexin 43 is a gap junction protein that plays a crucial role in intercellular communication. This antibody can be used in various research applications, such as immunohistochemistry and Western blotting, to detect and analyze the expression of Cx43 in biological samples.
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Lab products found in correlation
Rabbit anti aqp4, by Merck Group (1 mentions)
Rabbit anti cx30, by Thermo Fisher Scientific (1 mentions)
Cryostat, by Leica (1 mentions)
Enhanced chemiluminescence detection reagent, by Thermo Fisher Scientific (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Xcell 2 blot module, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg antibody, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Visionworks, by Analytik Jena (1 mentions)
Rabbit anti cx36, by Thermo Fisher Scientific (1 mentions)
0.2 μm polyvinylidene difluoridemembrane, by Bio-Rad (1 mentions)
Mouse anti tubulin, by Merck Group (1 mentions)
Biospectrum imaging system, by Analytik Jena (1 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit igg fitc sc 2090, by Santa Cruz Biotechnology (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Rabbit anti cx26, by Thermo Fisher Scientific (1 mentions)
Mitotracker green, by Thermo Fisher Scientific (1 mentions)
Ix81 microscope, by Olympus (1 mentions)
4 protocols using rabbit anti cx43
1
Immunofluorescence Staining of Cx43 in Mouse Fibroblasts
2
Immunohistochemical Analysis of Brain Tissue
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At 3 days, one part of the animals was transcardially perfused with 4% paraformaldehyde at 4 °C, brains were extracted and put in 30% sucrose before freezing, then stored at −20 °C. Coronal sections were cut at 20 μm thickness on a cryostat (Leica, Richmond, IL, USA) and mounted on slides for immunohistochemical analysis37 (link),38 (link). The primary antibodies used were: rabbit anti-AQP4 (1:300, Chemicon, Temecula, CA, USA) and mouse anti-AQP4 (1:100, AbD Serotec-BioRad, Hercules, CA, USA)39 ; rabbit anti-Cx43 (1:100, Invitrogen), rabbit anti-Cx30 (1:100, ThermoFisher Scientific), and chicken anti-glial fibrillary acid protein (GFAP, 1:1000, Millipore, Temecula, CA, USA). Secondary antibodies were used at 1:1000 as appropriate for each primary antibody (all secondary antibodies from Invitrogen). Negative control staining where the primary antibody or secondary antibody was omitted showed no detectable labelling.
3
Comparative Analysis of Cx36 and Cx43 Protein Levels
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Tissue samples were sonicated in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% Na deoxycholate, 50 mM Tris), and total protein was determined using BCA protein assays (Thermo-Scientific, Waltham, MA). Experiments were performed on the XCell II Blot Module (Invitrogen). From each sample, 20 μg of protein was loaded into a lane on a NuPage 4–12% Bis-Tris gel (Life Technologies, Carlsbad, CA), transferred to 0.2-μm polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), and processed with a blocking solution of 3% BSA and 5% nonfat dry milk in 1 × tris-buffered saline and Tween-20 (TBST). Rabbit anti-Cx36 (0.5 μl/ml; Invitrogen), rabbit anti-Cx43 (0.2 μl/ml; Invitrogen), and mouse anti-tubulin (1:10,000; Sigma-Aldrich, St. Louis, MO) were used as primary antibodies, and were visualized with horseradish peroxidase-conjugated anti-rabbit antibody (1:10,000; Invitrogen) or anti-mouse IgG antibody (1:10,000; Sigma-Aldrich). Signals were enhanced using enhanced chemiluminescence detection reagents (Thermo-Scientific) and detected on a BioSpectrum imaging system (UVP, Upland, CA). Densities were quantified using VisionWorks (UVP) and normalized relative to tubulin.
4
Immunofluorescence Staining of Cell Junctions
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Cells were grown in 24-well plates with glass coverslips on the bottom, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.2% Triton X-100/PBS for 3 min. Coverslips were incubated for 1 h with the following primary antibodies: mouse anti-α-tubulin (T5168, Sigma-Aldrich, Steinheim, Germany), rabbit anti-Cx43 (710700, Invitrogen, USA), mouse anti-Cx43 (610061, Transduction Laboratories, Lexington, KY, USA), rabbit anti-Cx26 (71-0500, Invitrogen, USA), rabbit anti-Cx30 (HPA014846, Sigma-Aldrich, Steinheim, Germany), then rinsed with 1% BSA/PBS and incubated with secondary goat anti-mouse IgG H&L (Cy5) (ab6563, Abcam Cambridge, UK) or with donkey anti-rabbit IgG (FITC) (sc-2090, Santa Cruz, CA, USA) for 30 min. The F-actin network was visualized using Alexa Fluor 594 phalloidin (Invitrogen, USA); coverslips were incubated with the dye for 30 min at 37°C. Coverglasses were attached using Vectashield Mounting Medium with DAPI (Vector Laboratories, CA, USA) and sealed with clear nail polish. MitoTracker Green (Invitrogen, USA) was used to stain mitochondria in live cells following the manufacturer’s instructions. Analysis was performed using the Olympus IX81 microscope with UPlanSApo 20x/0.85 OI or PlanApo N 60x/1.42 OI lens and the Orca-R2 digital camera with the fluorescence excitation system MT10 and XCELLENCE software.
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