Immunofluorescence was performed with either EBs or ESC-CMs. Cells were fixed with methanol at -20 ºC for 15min, followed by permeabilization with 0.05% Triton-100. The cells were blocked with 10% FBS for 30 min at room temperature and incubated in PBS containing one of the primary antibodies overnight at 4ºC and followed by secondary antibodies for 2 hours at room temperature. The primary antibodies included mouse monoclonal anti-α-Actinin (Sigma Aldrich, A-7811, USA), rabbit polyclonal anti-Nkx2.5 (Santa Cruz, sc-376565, USA), rabbit polyclonal anti-desmoplakin I/II (Santa Cruz, sc-33555, USA), rabbit polyclonal anti-N-cadherin (Santa Cruz, sc-31030, USA) or rabbit polyclonal anti-Connexin 43 (Abcam, ab-11370, USA), mouse monoclonal anti-Connexin 43 (Abcam, ab-79010, USA), rabbit polyclonal anti-MLC-2v (Abcam, ab-79935, USA). The second antibodies included Alexa fluor 488-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA, USA), Alexa fluor 594-conjugated anti-mouse IgG (Invitrogen, USA), Alexa fluor 488-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) or Alexa fluor 594-conjugated anti-rabbit IgG (Invitrogen, USA). After washing, the fluorescence images were taken by fluorescence inverted microscope or an Olympus IX81-FV1000 inverted multiphoton laser confocal microscope 24 (link).
Anti connexin 43
Manufactured by Abcam
Sourced in United States, Denmark
Anti-connexin 43 is a primary antibody that specifically recognizes the connexin 43 protein. Connexin 43 is a component of gap junctions, which are intercellular channels that allow the exchange of small molecules between neighboring cells. This antibody can be used in various immunodetection techniques to study the localization and expression of connexin 43 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti α actinin, by Merck Group (4 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Alexa fluor 488 conjugated anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Anti nkx2, by Santa Cruz Biotechnology (2 mentions)
Anti ctnt, by Abcam (2 mentions)
Anti cardiac troponin t, by Abcam (2 mentions)
Fluoview fv10i, by Olympus (2 mentions)
Anti fibronectin, by Abcam (2 mentions)
Anti vimentin, by Agilent Technologies (2 mentions)
Anti laminin, by Merck Group (2 mentions)
Anti sarcomeric alpha actinin α actinin, by Merck Group (2 mentions)
Anti cardiac troponin t ctnt, by Abcam (2 mentions)
Hoechst 33342, by Dojindo Laboratories (2 mentions)
Anti occludin, by Abcam (2 mentions)
Anti n cadherin, by Abcam (2 mentions)
Anti zo 1, by Abcam (2 mentions)
Anti claudin 11, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti connexin 40, by Abcam (2 mentions)
15 protocols using anti connexin 43
1
Immunofluorescence Characterization of Cardiac Cells
2
Immunofluorescence Characterization of Cardiomyocytes
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Cells grown on chamber slides (BD) were fixed (paraformaldehyde 4% in PBS, 15 min). Every following step was performed in PBS containing 1% BSA (Sigma). Cells were permeabilised (0.1% Triton X-100, 25 min) and blocked with 5% normal goat serum (NGS, 15 min). Primary antibody incubation was performed at 4°C overnight, dilution 1 : 200. Primary antibodies, anti-HuNu (Millipore), anti-cTnT (Abcam), anti-Mef2c (New England Biolabs), anti-connexin 43 (Abcam), and anti-alpha-sarcomeric actinin (Abcam), were washed and samples were incubated with secondary antibodies (1 : 500) for 30 minutes at RT. Secondary antibodies used were as follows: anti-rabbit-Alexa488, anti-mouse-Alexa594, and anti-rabbit-Alexa555 (Invitrogen) and anti-mouse Alexa555 (Cell Signaling).
Stained specimens were kept in mounting media (PermaFluor, ThermoScientific) and images were acquired using a fluorescent microscope (EVOS, Life Technologies) or spinning disk confocal microscope (Quorum Zeiss AxioVert, SickKids Imaging Facility, Toronto). Images were quantified using Volocity™ (Perkin-Elmer) imaging software.
Stained specimens were kept in mounting media (PermaFluor, ThermoScientific) and images were acquired using a fluorescent microscope (EVOS, Life Technologies) or spinning disk confocal microscope (Quorum Zeiss AxioVert, SickKids Imaging Facility, Toronto). Images were quantified using Volocity™ (Perkin-Elmer) imaging software.
3
Immunofluorescence Staining of Cells
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The cells were fixed with 4% formalin for 10 min at room temperature and washed 4 times with PBS. To permeabilize the cell membrane, the samples were incubated in cold (−20 °C) 90% methanol for 5 min at 4 °C. Methanol was decanted and the cells were rinsed 3 times with PBS. The samples were treated with a blocking solution of 5% FBS in PBS at 37 °C for 60 min. While blocking, the dilution of primary monoclonal antibodies were prepared in 5% FBS. The 500–600X dilutions of anti-connexin 43, anti-cardiac troponin T or anti-CD31 primary antibodies (Abcam) were used. The blocking solution was removed and the samples were incubated with the solution of primary antibodies at 4 °C for 12 h. The solution was aspirated and the samples were washed 3 times with PBS. Following that, the samples were incubated in solution 900X-diluted FITC- and TRITC-conjugated secondary antibodies in the dark for 2 h at room temperature. The samples were washed 3 times with PBS and visualized under fluorescent microscope.
4
Cardiac Troponin T and Connexin 43 Visualization
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The cells were fixed in 4 °C acetone for 15 min and blocked with goat serum (1:20). Then, primary anti-cardiac troponin T monoclonal (1:400, Abcam) and anti-connexin 43 polyclonal (1:100, Abcam) antibodies were added for overnight incubation at 4 °C. Next, secondary antibodies (1:150; CoWin Bioscience, Beijing, China) conjugated to Cy3 were added and incubated for 1 h at 37 °C. DAPI was then added for 3 min. Images were acquired under a fluorescence microscope (BX51; Olympus).
5
Immunofluorescence Staining of Cell-Scaffold Constructs
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For immunofluorescence staining, the cell-scaffold constructs were fixed in 4% formaldehyde for 30 minutes at room temperature, permeabilized with 0.3% Triton X-100 in PBS for 10 minutes, and then blocked with 2% BSA, 0.05% sodium azide in PBS for 45 minutes to block nonspecific antibody binding. Subsequently, constructs were incubated with the primary antibodies overnight at 4°C. The primary antibodies used were as follows: rabbit monoclonal anti-Troponin I (1:100; AbCam, Cambridge, MA, USA), and mouse monoclonal anti-α-SA (1:100; AbCam), rabbit polyclonal anti-connexin-43 (1:1,000; AbCam), rabbit polyclonal anti-GATA-4 (1:100; Santa Cruz, CA, USA) and rabbit polyclonal anti-Nkx2.5 (1:200; Santa Cruz, CA, USA). Constructs were then washed three times for 10 minutes with PBS and incubated with Alexa Fluor 488- or Alexa Fluor 548-conjugated secondary antibodies (1:500; Invitrogen) for 1 hour in the dark at room temperature. Confocal images were acquired using a Zeiss LSM 510 microscope. Immunofluorescent staining was also performed to assess cell proliferation on the scaffolds. On days 1 and 3 of culture, the cell-scaffold constructs were stained with antibodies against proliferating cell nuclear antigen (PCNA; 1:200; Abcam). Twenty different images were randomly captured for each group, and the PCNA-positive cells were quantified using ImageJ software.
6
Connexin 43 Expression in hiPSC-CMs
7
Connexin 43 Expression Analysis
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Protein was extracted from the TA and SOL muscles in the CON and DEN groups. Western blot analysis was performed as previously described [25 (link)]. The primary antibodies used in this study were as follows: anti-connexin 43 (abcam) and anti-GAPDH (Cell Signaling Technology). The band intensities of the target proteins were analyzed using the National Institutes of Health Image J software and normalized by the band intensity of GAPDH.
8
Immunofluorescence Characterization of hiPSC-Derived Cardiomyocytes
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hiPSC-CMs and 3D-hiPSC-CT were fixed with 4% paraformaldehyde. hiPSC-CMs and 3D-hiPSC-CT were labeled by primary antibodies such as anti-cardiac troponin T (cTnT, 1:200 dilution; Abcam, Cambridge, UK), anti-sarcomeric alpha actinin (α-actinin, 1:400; Sigma), anti-vimentin (1:100; Dako, Glostrup, Denmark), anti-connexin43 (1:100; Abcam), anti-MLC2a (1:100; Synaptic Systems GmbH), anti-MLC2v (1:200; Proteintech, Rosemont) anti-fibronectin (1:200; Abcam), anti-laminin (1:30; Sigma), anti-collagen type I (1:100; Abcam), or anti-collagen type III (1:100; Abcam), followed by secondary antibodies such as AlexaFluor488 or AlexaFluor555 conjugated goat or donkey anti-mouse or anti-rabbit (ThermoFisher Scientific). Nuclei were counterstained with Hoechst33342 (Dojindo, Kumamoto, Japan) and assessed using confocal microscope (FLUOVIEW FV10i; Olympus, Tokyo, Japan).
9
Immunohistochemical Analysis of Testicular Tight Junctions
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The IHC experiment was performed as described previously [62 (link)]. Briefly, testes sections (6 μm) were dewaxed with xylene and gradient ethanol consecutively, followed by antigen retrieval. The sections were incubated with 0.5% bovine serum albumin for 1 h and then incubated overnight with the following antibodies: anti-ZO-1 (1:100, Cat# ab276131, Abcam, Cambridge, MA, USA), anti-Occludin (1:200, Cat# ab216327, Abcam, Cambridge, MA, USA), anti-Claudin-11 (1:100, Cat# 36-4500, Thermo Fisher Scientific, Waltham, MA, USA), anti-Connexin-43 (1:500, Cat# ab217676, Abcam, Cambridge, MA, USA), and anti-N-cadherin (1:500, Cat# ab207608, Abcam, Cambridge, MA, USA). The sections were incubated for 1 h with HRP-labeled secondary antibody (1:2000, Cat# ab205722, Abcam, Cambridge, MA, USA), followed by DAB staining (Cat# ab64264, Abcam, Cambridge, MA, USA) and observation under a microscope (Nikon, Tokyo, Japan).
10
Immunofluorescence Staining of Schwann Cells
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IF staining of SCs was conducted according to a previous study [66 (link)]. Briefly, SCs were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 15 min, and blocked with 1% BSA for 1 h. Then, SCs were incubated with anti-WT1 (1:50, Cat# ab267377, Abcam, Cambridge, MA, USA), anti-Vimentin (1:100, Cat# ab92547, Abcam, Cambridge, MA, USA), anti-ZO-1 (1:100, Cat# ab276131, Abcam, Cambridge, MA, USA), anti-Occludin (1:100, Cat# ab216327, Abcam, Cambridge, MA, USA), anti-Claudin-11 (1:100, Cat# 36-4500, Thermo Fisher Scientific, Waltham, MA, USA), anti-Connexin-43 (1:100, Cat# ab217676, Abcam, Cambridge, MA, USA), anti-N-cadherin (1:100, Cat# ab207608, Abcam, Cambridge, MA, USA), and anti-LC3B (1:200, Cat# ab192890, Abcam, Cambridge, MA, USA) overnight at 4 °C. Then, the SCs were incubated with secondary antibody IgG H&L (1:1000, Cat# ab150073, Abcam, Cambridge, MA, USA) at room temperature for 1 h, followed by the nuclei staining of DAPI (Cat# ab104139, Abcam, Cambridge, MA, USA) for 10 min. The images were captured by a fluorescence microscope (Olympus BX53, Tokyo, Japan).
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