Hearts from WT, mdx, and mdx:utr mice were dissected out, rinsed in sterile PBS, and frozen in O.C.T. using liquid nitrogen cooled isopentane. Human DMD heart samples were similarly frozen in O.C.T. using liquid nitrogen cooled isopentane. Cryosections were cut at 6 μm and stained with an antibody reactive to Cx43 (anti-mouse, Sigma, C8093, 1:500) along with a counterstain of wheat germ agglutinin (WGA, Invitrogen, 1:1000) to demarcate cell borders or N-cadherin (anti-rat DSHB, MNCD2, 1:20). Slides were coverslipped with a mounting medium containing DAPI and sections were imaged and analyzed using a Nikon A1R confocal (60 × objective, Fig. 1 ) or Eclipse T1 (40 × objective, Fig. 2 ) microscope and NIS-Elements AR software.
C8093
Manufactured by Merck Group
The C8093 is a laboratory instrument designed for conductivity measurement. It provides accurate and reliable conductivity readings for various applications in a laboratory setting. The core function of this product is to measure the electrical conductivity of solutions, which is a fundamental property used in numerous scientific and analytical procedures.
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Lab products found in correlation
A1r confocal, by Nikon (1 mentions)
Wheat germ agglutinin (wga), by Thermo Fisher Scientific (1 mentions)
Cy3 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Mouse monoclonal anti plakoglobin, by Merck Group (1 mentions)
P8087, by Merck Group (1 mentions)
T9026, by Merck Group (1 mentions)
Amersham ecl plus western blotting detection kit, by GE Healthcare (1 mentions)
Amersham ecl, by Cytiva (1 mentions)
Envision detection system, by Agilent Technologies (1 mentions)
Ab34710, by Abcam (1 mentions)
A7732, by Merck Group (1 mentions)
Ab16502, by Abcam (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Gs 800 calibrated imaging densitometer, by Bio-Rad (1 mentions)
T4026, by Merck Group (1 mentions)
G9023, by Merck Group (1 mentions)
C1 confocal microscope, by Nikon (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
7 protocols using c8093
1
Quantifying Cx43 and Cell Borders in Mouse and Human Heart
2
Immunolabeling of Cell-Cell Junctions
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Buccal mucosa smears were immunostained with the following primary antibodies using established protocols: mouse monoclonal anti-plakoglobin (P8087, Sigma Aldrich), mouse monoclonal anti-plakophilin-1 (325700, Thermo Fisher Scientific), mouse monoclonal anti-desmoplakin (10R-D108AX, Fitzgerald), and mouse polyclonal anti-Cx43 (C8093, Sigma Aldrich). Slides were then incubated with Cy3-conjugated secondary antibodies (Jackson ImmunoResearch) for 2 h at room temperature and counterstained with DAPI to label nuclei. All immunostained preparations were imaged at x20 using a Nikon A1R confocal microscope.
We optimized our immunostaining protocol for each protein by first determining the lowest primary antibody concentration that produced strong signal in control buccal mucosa cells. These conditions were then applied throughout the study to determine whether cells from subjects from ACM families showed apparent reduction in junctional signal. Using this approach, we consistently observed either control levels of signal or a virtual absence of junctional signal for any given primary antibody. This ‘binary’ approach ensures reproducibility of our results and precludes the need for signal quantification. Each batch of ACM samples was immunostained alongside freshly-obtained smears from age-matched controls.
We optimized our immunostaining protocol for each protein by first determining the lowest primary antibody concentration that produced strong signal in control buccal mucosa cells. These conditions were then applied throughout the study to determine whether cells from subjects from ACM families showed apparent reduction in junctional signal. Using this approach, we consistently observed either control levels of signal or a virtual absence of junctional signal for any given primary antibody. This ‘binary’ approach ensures reproducibility of our results and precludes the need for signal quantification. Each batch of ACM samples was immunostained alongside freshly-obtained smears from age-matched controls.
3
Western Blot Analysis of Connexin 43
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Cells were harvested using lysis buffer (Tris•HCl pH 7.4 20 mM, NaCl 150 mM, EDTA 1 mM, EGTA 1 mM, Triton 1%, sodium pyrophosphate 2.5 mM, Na3VO4 1 mM, β-glycerophosphate 1 mM, protease inhibitors 1:1000). Monoclonal antibodies against Cx43 (Sigma, C8093, 1:5000) or α-tubulin (Sigma, T9026, 1:10000) were used. Immunopositive bands were visualized by Amersham ECL™ Plus Western Blotting Detection Kit (GE Healthcare), and were quantitatively analyzed using image J software.
4
Immunohistochemical Profiling of Cartilage Markers
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The paraffin-embedded specimens were cut 4-μm thickness. Briefly, following sections were deparaffinized and rehydrated. The antigen retrieval step was performed by microwave heating base using EDTA buffer (pH=9) for all markers except NF-κB. For NF-κB citrate buffer (pH=6) was used. The endogenous peroxidase was blocked by 0.3% H2O2 and protein blocking was done by 2% bovine serum albumin mixed in normal sheep serum. After that, the slides were incubated with a primary antibody to collagen I monoclonal antibody [Col I], (ab34710, Abcam, 1:750), col II, [CP18], (Sigma-Aldrich,1:150), NF-κB, (ab16502, Abcam, 1:500), anti-mitochondria, (MAB1273, Merk, 1:150), anti-α-Actinin (Sarcomeric) (A7732, Sigma-Aldrich, 1:100), and connexin-43, (C8093, Sigma-Aldrich, 1:100) at 4°C for overnight. The Envision detection system (Dako K5007-Denmark) was also used. Finally, the slides were counterstained with Mayer's hematoxylin (Bahar afshan-Iran) for 2 min, dehydrated, and mounted and analyzed by a veterinary anatomic pathologist. The quantitative evaluation of NF-κB expression was performed using ImageJ software (ImageJ, NIH, Bethesda, MD, USA).
5
Western Blot Analysis of CX45, NANOG, and CX43
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Cell lysates were separated via SDS–PAGE using 8% (w/v) Tris-glycine mini-gels and transferred to polyvinylidene difluoride membranes as previously described46 (link). The primary antibodies used were as follows: rabbit anti-CX45 (1:1 000; MAB3100; Millipore), rabbit anti-NANOG (1:2 000; D73G4; Cell Signaling), mouse anti-CX43 (1:2000; C8093; Sigma), and mouse anti-beta-TUBULIN (1:10 000; T4026; Sigma). After incubation with enhanced chemiluminescence (Amersham), immunopositive bands were visualized and scanned with a GS-800 Calibrated Imaging Densitometer (Bio-Rad). All of the western blotting exposures were within the linear detection range. The intensity (area × density) of the individual bands on western blotting was measured by a Quantity-One software (Bio-Rad) and the amount of target protein was normalized to β-TUBULIN.
6
Immunohistofluorescence of Connexin43 in Uterus
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Immunohistofluorescence was used to localise Cx43 in uterine tissues. Cross-sections of uterine horn samples were fixed in 4% PFA in 0.1 M PBS (pH 7.4), and cryoprotected in 18% sucrose. Immunostaining was carried out on consecutive 7 μm cryostat sections. To block endogenous peroxidase, the sections were treated with hydrogen peroxide in methanol and washed in 0.1 M PBS. The sections were blocked with 10% normal donkey serum (Sigma, G9023) for 1 h at room temperature (approx. 23 o C; RT), and incubated overnight at RT with a 1:500 dilution of anti-Cx43 (Sigma, C8093), Next, the cells were washed 3x with PBS and incubated 1 h at RT with secondary antibodies conjugated with cyanine 3 (CY 3 ; Jackson ImmunoResearch, West Grove, PA, 715-165-150). Connexin43 was visualized with confocal imaging using a Nikon C1 confocal microscope.
7
Western Blot Analysis of Cx43 Protein
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Protein expression for Cx43 in the tissues and cells was determined by Western blotting. Proteins from homogenized tissues and in vitro cultured cells were released with lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% TritonX-100, 0.5% sodium deoxycholate and protease inhibitors (Sigma, P8340). The lysates were stored at -86 o C until further analysis. Protein concentrations were measured by the Bradford's method.
Western blot analysis was performed as previously described (Korzekwa et al. 2011) . Equal amounts of protein were dissolved in SDS gel-loading buffer, heated to 95 o C for 4 min and separated in 10% SDS-PAGE gel. Separated proteins were electrob- lotted onto 0.2 μm nitrocellulose membranes in transfer buffer. After blocking in 5% non-fat dry milk in TBS-T buffer for 1.5 h at RT, the membranes were incubated overnight with a 1:500 dilution of anti-Cx43 (Sigma, C8093) antibodies; GAPDH (Sigma, G8795; monoclonal anti-glyceraldehyde-3--phosphate dehydrogenase antibody produced in mouse) expression was used as a reference. Proteins were detected by incubating the membranes with a 1:20,000 dilution of secondary polyclonal anti-mouse alkaline phosphatase-conjugated antibody (Sigma, A 3562) for 1.5 h at RT. Western blots were quantitated using the Kodak 1 D program (Eastman Kodak, Rochester, NY, USA).
Western blot analysis was performed as previously described (Korzekwa et al. 2011) . Equal amounts of protein were dissolved in SDS gel-loading buffer, heated to 95 o C for 4 min and separated in 10% SDS-PAGE gel. Separated proteins were electrob- lotted onto 0.2 μm nitrocellulose membranes in transfer buffer. After blocking in 5% non-fat dry milk in TBS-T buffer for 1.5 h at RT, the membranes were incubated overnight with a 1:500 dilution of anti-Cx43 (Sigma, C8093) antibodies; GAPDH (Sigma, G8795; monoclonal anti-glyceraldehyde-3--phosphate dehydrogenase antibody produced in mouse) expression was used as a reference. Proteins were detected by incubating the membranes with a 1:20,000 dilution of secondary polyclonal anti-mouse alkaline phosphatase-conjugated antibody (Sigma, A 3562) for 1.5 h at RT. Western blots were quantitated using the Kodak 1 D program (Eastman Kodak, Rochester, NY, USA).
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