Frozen sections of heart tissue (14 days after adenoviral transduction) were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 and then incubated with the appropriate primary antibody—sarcomeric α-actinin (Abcam, ab9465, 1:200), Cx43 polyclonal antibody (Sigma, C6219, 1:100), ZsGreen polyclonal antibody (Clontech, 632474, 1:200) for TBX18 group, and GFP antibody (Abcam, ab290, 1:200) for GFP group—and Alexa Fluor–conjugated secondary antibodies (Invitrogen). Counterstaining with DAPI (Molecular Probes) was performed. Sections were imaged using a confocal laser scan microscope (Leica Microsystems), and images were processed by Leica LAS software suite.
Sarcomeric α actinin
Manufactured by Abcam
Sourced in United States, United Kingdom
Sarcomeric α-actinin is a structural protein found in the Z-discs of skeletal and cardiac muscle. It plays a key role in the organization and stability of the sarcomere, the basic contractile unit of muscle cells.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescently labeled secondary antibody, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Laminin, by Abcam (2 mentions)
Hoechst 33258, by Dojindo Laboratories (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ab9465, by Merck Group (1 mentions)
Confocal laser scan microscope, by Leica (1 mentions)
Ab290, by Abcam (1 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
Collagen 3, by Abcam (1 mentions)
Ssea4, by Abcam (1 mentions)
Oct3 4, by Santa Cruz Biotechnology (1 mentions)
Fibronectin, by Abcam (1 mentions)
Nanog, by Santa Cruz Biotechnology (1 mentions)
Cm1850 cryostat, by Leica (1 mentions)
Fv1200 microscope, by Olympus (1 mentions)
Alexa fluor 633, by Thermo Fisher Scientific (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Phospho pkcα, by Cell Signaling Technology (1 mentions)
8 protocols using sarcomeric α actinin
1
Cardiac Tissue Immunofluorescence Analysis
2
Pluripotent Stem Cell and Cardiomyocyte Immunostaining
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Cells were fixed in 4% paraformaldehyde and permeabilized with 0.05% Triton-X100 followed by goat serum blocking. H7 and hiPSC colonies were stained with pluripotency marker antibodies OCT3/4 (Santa Cruz), SOX2 (Abcam), Nanog (Santa Cruz), and SSEA-4 (Abcam), whereas hPSC-derived cardiomyocytes were stained with antibodies for cTNT (Abcam), Sarcomeric α-actinin (Abcam), MYL2 (Proteintech), MYL7 (Synaptic system), or EGFP (Proteintech) for 24 h at 4 °C respectively. Cells were then incubated with Alexa Fluor 594 or 488 at 37 °C for 1 h and subsequently counterstained with DAPI. For rat hearts, heart tissues were paraffin-embedded and sectioned, followed by H&E staining. The remaining tissues were embedded with optimal cutting temperature compound (OCT; Sakura Finetek, Japan) and sectioned into sections 8 mm thick. The slides were then labeled with antibodies for Laminin (Thermo Fisher Scientific), fibronectin (Abcam), and collagen III (Abcam). Images were captured with a fluorescence microscope Leica DMi8 (Leica).
3
Localization of Phospho-PKC Isoforms
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Frozen ventricular tissue sections (4 µm) were prepared using cryostat CM1850 (Leica, CA) from all experimental groups. Tissue sections were fixed and stained with antibodies against phospho-PKC-δ and phospho-PKC-α (Cell signaling) and sarcomeric α-actinin (Abcam), followed by incubation with labeled secondary antibodies Alexafluor 488, and Alexa fluor 633 (Molecular Probes, OR) as described earlier [17] (link). After mounting with Vectashield [with DAPI] (Vector Laboratories, CA), tissue sections were visualized under confocal FV1200 microscope (Olympus, PA).
4
Immunostaining of Myofiber Constructs
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Cells encapsulated in the Fib/Mtr gel were fixed with 2% paraformaldehyde in PBS overnight at 4 °C. Following fixation, samples were washed with phosphate buffered saline (PBS) and then incubated in blocking solution (5% bovine serum albumin with 0.2% Triton-X 100) (Sigma-Aldrich) for 12 h30 (link),31 (link). The myofiber sheet with Fib/Mtr gel was treated with primary antibodies (sarcomeric α-actinin (1:500) (Abcam, Cambridge, MA, USA), laminin (1:500) (Abcam), type IV collagen (1:500) (Abcam), myosin heavy chain (1:200) (R&D Systems, Minneapolis, MN, USA), myogenin (1:500) (Thermo Fisher Scientific)) or AlexaFluor 568-conjugated phalloidin at 4 °C overnight. After washing with PBS, the tissue constructs were treated with fluorescently labeled secondary antibodies (1:800) (Thermo Fisher Scientific) for 2 h at 37 °C. For nuclei staining, tissue constructs were incubated with Hoechst33258 (Dojindo Laboratories, Kumamoto, Japan) for 5 min at RT. Images were acquired using a Zeiss 510 inverted confocal microscope and analyzed using LSM Image Software.
5
Cardiomyocyte Immunofluorescence Staining
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Cells were seeded on Matrigel-coated coverslips and were left to attach for 2 days. Cells were washed with 3 X PBS, fixed with 4% parafomaldehyde (15 mins), washed and permabilized with 0.2% Triton-X (15 mins), washed and blocked for 3 hours in 3% goat serum and incubated with cTnT (Abcam) and sarcomeric α-actinin (Abcam) overnight. Cells were washed with 3 X PBS and incubated for one hour with secondary antibody Alexa Fluor-563 (Abcam) and Alexa Fluor-488 (Abcam) and were then washed and mounted on a slide using Antifate mounting medium containing DAPI (VECTASHIELD, VECTOR laboratories). Slides were viewed on Zeiss LSM880 confocal microscope.
6
Immunohistochemistry of Cardiac Tissue
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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.
7
Reagents for Cell Culture and Immunohistochemistry
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Py was obtained from Sigma-Aldrich Corporation (USA). Other chemical reagents were obtained from Guanghua Chemical Factory (China). Dulbecco’s modified Eagle’s medium (DMEM)/high-glucose medium, fetal bovine serum (FBS), and trypsin were purchased from Gibco (USA). The primary antibodies including CD68, Ki-67, vWF, α-smooth muscle actin (α-SMA), sarcomeric α-actinin, and CX-43 were ordered from Abcam (Britain). Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L) and Alexa Fluor 568 Donkey Anti-Mouse IgG (H+L) were procured from Life Technologies (USA).
8
Immunofluorescent Imaging of Myofiber Sheets
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Myofiber sheet tissues were fixed with 2% paraformaldehyde in PBS overnight at 4°C. After fixation, samples were washed with PBS and then incubated in blocking solution (5% bovine serum albumin with 0.2% Triton-X 100; Sigma-Aldrich) for 10 h. 26 The coculture tissues were washed with PBS, and then treated with primary antibodies (neurofilament [NF; 1:500], islet [1:500], sarcomeric α-actinin [1:500], laminin [1:500]; Abcam, Cambridge, MA) at 4°C overnight. After being washed with PBS, the tissues were treated with fluorescently labeled secondary antibodies (1:800; Thermo Fisher Scientific) or Alexa Fluor 488-conjugated α-bungarotoxin (Thermo Fisher Scientific) at RT for 2 h. For nuclei staining, tissues were incubated with Hoechst33258 (Dojindo Laboratories, Kumamoto, Japan) at RT for 2 h. Images were acquired using a confocal laser microscope (FV1200; Olympus, Tokyo, Japan). To measure the diameter of myofibers, immunofluorescence images of α-actinin-positive myofibers were taken under five representative fields, and all data were expressed as mean ± standard deviation (n = 3).
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