Samples for immunohistochemistry were either fixed in PFA and paraffin-embedded or equilibrated through a sucrose series (to 15%) and subsequently mounted and frozen in OTC (Tissue-tek). In the latter case, air-dried sections were fixed with methanol or 4% PFA for immunostaining. Primary antibodies utilized were Scribble (Santa Cruz); Vangl2 (gift from Dr Charlotte Dean, London, UK), MF20 (DSHB), phospho-histone H3 (Millipore), cleaved caspase-3 (Cell Signalling), sarcomeric α actinin (Abcam), alpha-smooth muscle actin (α-SMA) (Sigma), cardiac troponin I (Hytest Ltd), Rac1 (Millipore), β-PIX (Millipore), β-catenin (BD Transduction Laboratories), N-cadherin (BD Transduction Laboratories), and connexin-43 (Chemicon). Alexa fluor 488 and 596-conjugated secondary antibodies (Invitrogen) were used to detect the primary antibody. Phalloidin (Sigma) was used to stain the actin cytoskeleton and wheat germ agglutinin (Alexa fluor 647; Invitrogen) was used to stain cell membranes. Cell nuclei were identified using DAPI. Immunofluorescence images were collected with using a Zeiss Axioimager Z1 fluorescence microscope equipped with Zeiss Apotome 2 (Zeiss, Germany). Acquired images were processed with the AxioVision Rel 4.9 software.
Connexin 43
Manufactured by Merck Group
Sourced in United States
Connexin 43 is a laboratory equipment product manufactured by Merck Group. It is a protein involved in the formation of gap junctions, which facilitate intercellular communication. This product is used for research purposes to study cell-to-cell interactions and signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
β catenin, by BD (2 mentions)
α actinin, by Merck Group (2 mentions)
Connexin 26, by Santa Cruz Biotechnology (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Apotome 2, by Zeiss (1 mentions)
Sarcomeric α actinin, by Abcam (1 mentions)
β pix, by Merck Group (1 mentions)
Phospho histone h3, by Merck Group (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
N cadherin, by BD (1 mentions)
Alpha smooth muscle actin α sma, by Merck Group (1 mentions)
Cardiac troponin 1, by HyTest (1 mentions)
Cleaved caspase 3, by Merck Group (1 mentions)
Sytox green nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
Fluoro gel, by Electron Microscopy Sciences (1 mentions)
Tuj1 mms 435p, by Fortrea (1 mentions)
Pax6 prb 278p, by Fortrea (1 mentions)
19 protocols using connexin 43
1
Immunohistochemistry of Cardiac Tissue
2
Immunofluorescence Staining of Tissue Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cryostat sections were incubated with blocking buffer (2.5% BSA, 0.01% Triton-X100) for 1 h at RT and incubated with primary antibody diluted in blocking buffer overnight at 4°C. Sections were washed with PBS and incubated with secondary antibody for 2 h at RT, washed with PBS and mounted using Fluoro-Gel (Electron Microscopy Services #17985-11). Antibodies used were: Abcam: CTIP2 (ab18465) 1:300; BD Transduction Labs: N-cadherin (610920) 1:500; Cell Signaling Technology: Cleaved caspase-3 (#9661) 1:200, Connexin-43 (#3512) 1:300, Ki67 (#9129) 1:400, phosH3 (#9706S) 1:300; Chemicon: Sox2 (AB5603) 1:400, MAP2 (MAB3418) 1:400, TBR2 (AB9618) 1:400; Covance: Tuj1 (MMS-435P) 1:400, Pax6 (PRB-278P) 1:400. The Pax6 mAb (1:200) developed by A. Kawakami was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Sections were incubated in the relevant secondary antibody conjugated to Alexa-Fluor 488 nm, 568 nm or 647 nm (Molecular Probes/Invitrogen) and counter-stained with DAPI (4’,6-diamidino-2-phenylindole) and/or SYTOX® green nucleic acid stain (Molecular Probes/Invitrogen), prior to mounting with Fluoro-Gel.
3
Histological Analysis of Mouse Hearts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Once the physiological measurements were complete, the mice were sacrificed. Hearts were removed, weighed, and cut into transverse slices at the level of the mid-papillary muscle. The slices were then fixed in 4% paraformaldehyde, embedded in paraffin, cut into 5-μm thick sections, and stained with hematoxylin and eosin29 (link).
For cryosections (5 μm), mouse hearts were equilibrated in 30% sucrose solution and mounted in FSC 22® frozen section compound. (Leica, Wetzlar, Germany). Sections were fixed with ethanol at RT for 10 min, subjected to immunofluorescence staining with primary antibodies against connexin 43 (1:500; Sigma-Aldrich) and N-cadherin (1:500 dilution, Chemicon, Temecula, CA, USA), and then visualized with secondary fluorescent antibodies. Sections were counterstained with Hoechst33342 and examined on a Zeiss Axioskop2 Plus microscopy system (Carl Zeiss, Oberkochen, Germany). Photomicrographs were captured using an AxioCam HRc digital camera.
For cryosections (5 μm), mouse hearts were equilibrated in 30% sucrose solution and mounted in FSC 22® frozen section compound. (Leica, Wetzlar, Germany). Sections were fixed with ethanol at RT for 10 min, subjected to immunofluorescence staining with primary antibodies against connexin 43 (1:500; Sigma-Aldrich) and N-cadherin (1:500 dilution, Chemicon, Temecula, CA, USA), and then visualized with secondary fluorescent antibodies. Sections were counterstained with Hoechst33342 and examined on a Zeiss Axioskop2 Plus microscopy system (Carl Zeiss, Oberkochen, Germany). Photomicrographs were captured using an AxioCam HRc digital camera.
4
Isolation and Culture of Human Pulmonary Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human pulmonary artery endothelial cells (HPAECs), human pulmonary microvascular endothelial cells (PMVECs), and cell culture medium were obtained from Lonza Inc. (Allendale, NJ, USA), and cells were used at passages 5–8. Di-Phospho-MLC antibodies were obtained from Cell Signaling (Beverly, MA, USA). Beta-actin and Connexin 43 antibodies were obtained from Sigma (St Louis, MO, USA). Unless specified, biochemical reagents were obtained from Sigma.
5
Western Blot Analysis of Connexin 43 and EMT Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed as previously described. First, 100 μg of protein was separated by 10% SDS–PAGE gel and transferred to PVDF membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 2 hours and then were incubated with primary antibodies at 4°C overnight. The primary antibodies included Connexin 43 (C6219, Sigma Aldrich) and EMT sampler kit. Horseradish peroxidase-conjugated secondary antibodies were used and the protein bands were visualized by an enhanced chemiluminescence detection system (Amersham Bioscience, Piscataway, NJ, USA) according to the manufacturer’s protocol.
6
Immunohistochemical Analysis of Cell-Cell Junctional Proteins in Human Optic Nerve
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Optic nerve samples including the bulbar, midorbital, and intracanalicular segments were formalin fixed and paraffin embedded. For each segment of human ON samples, 4 μm coronal and sagittal sections were prepared using a microtome. Slides with ON sections were stained with the following antibodies: junctional adhesion molecule A (Novus Biologicals, H00050848-M01), Occludin (abcam, ab31721), Claudin 5 (abcam, ab15106), Connexin 43 (Sigma, C6219), Connexin 26 (abcam, ab38584), and Desmoplakin I + II (Progen, 65146). Antigen retrieval using CC1 buffer (Tris/Borate/EDTA buffer, pH 8.0–8.5) was performed for all antibodies. The extent of cell–cell interaction markers was semiquantitatively determined by eye (scale ranging from 1 to 3). To evaluate specificity of immunohistochemistry staining procedure, control staining (absence of primary antibody) for each marker was performed on coronal sections of human meninges (retrobulbar portion of the ON) (Figure S1 in Supplementary Material).
7
Immunocytochemistry of Tight Junctions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunocytochemistry was also performed on ARPE-19 cells grown as monolayers on transwell plates, fixed in 4% paraformaldehyde. After extensive washing, cells were incubated either in rabbit polyclonal antibodies recognizing ZO-1 (1:200; Invitrogen, Carlsbad, CA, USA), occludin (1:200; Invitrogen) or connexin43 (1:300; Sigma Aldrich, St Louis, MO, USA) in blocking solution (10% normal goat serum and 0.4% Triton-X in tris-buffered saline), and followed by Alexa Fluor 488 goat-anti-rabbit (1:500; Invitrogen) as the secondary antibody. All immunohistochemistry experiments included a no-primary antibody control (data not shown). Staining was examined via fluorescence microscopy (Zeiss, Thornwood, NY, USA) equipped with a digital black-and-white camera (Spot camera; Diagnostic Instruments, Sterling Heights, MI, USA).
8
Western Blot Analysis of Connexin-43
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LV tissue samples were snap-frozen in liquid nitrogen and stored at −80°C. Proteins (50 µg) were separated on acrylamide gels (TGX Stain-Free Precast Gels; Bio-Rad) and transferred (Trans-Blot Turbo; Bio-Rad) onto polyvinylidene difluoride membranes. After incubation with primary antibodies (Connexin-43, 1:500; Sigma-Aldrich) and HRP-conjugated secondary antibodies (1:2000; Bio-Rad), total hybridized protein levels were imaged under UV light and immunospecific signals revealed by enhanced chemiluminescence (Thermo Scientific). Quantification of Western blot was done with ImageJ (National Institutes of Health).
9
Quantitative Analysis of Cardiomyocyte Maturation
10
Immunoblot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysate was prepared using lysis buffer consisting of 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.5% deoxycholate, 1% NP-40, 0.3% SDS, and 1 mM phenylmethylsulfonyl fluoride.29 (link) Immunoblot analyses were performed according to the procedure described previously.29 (link) The antibodies used were specific for human STAT3, phosphorylated STAT3 (EMD Millipore, Billerica, MA, USA), HSP90α (AbD Serotec), phosphorylated FAK (Invitrogen), phosphorylated CDK2 (Epitomics, Burlingame, CA, USA), phosphorylated Rb, Rb, phosphorylated CDC2 (Cell Signaling, Danvers, MA, USA), connexin-43 (Sigma, St. Louis, MO, USA), integrin αV (BD Biosciences, San Jose, CA, USA), cyclin D1, MMP-2, MMP-9 (Lab Vision/NeoMarkers Co., Fremont, CA, USA), CDK4, cyclin E, cyclin A, CDK2, cyclin B1, CDC2, FAK, TCF12, Twist-1, Snail, Slug, E-cadherin, connexin-26, fibronectin, and GAPDH (Santa Cruz Biotechnology).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!