Human cardiomyocyte immunocytochemistry kit

Manufactured by Thermo Fisher Scientific

The Human Cardiomyocyte Immunocytochemistry Kit is a laboratory tool designed for the detection and visualization of cardiomyocyte-specific proteins in human cell samples. The kit provides the necessary reagents and protocols to perform immunocytochemistry assays.

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Cardiomyocyte Immunocytochemistry Kit (2 citations)

6 protocols using human cardiomyocyte immunocytochemistry kit

Immunocytochemistry of Pluripotent and Cardiac Cells

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Undifferentiated hESCs were plated on vitronectin-coated coverslips and processed for immunofluorescence 72 h after seeding. Briefly, cells were fixed for 12 min with 3% paraformaldehyde at room temperature, permeabilized with 0.25% Triton X-100 in PBS, incubated overnight with primary antibodies in presence of blocking solution, washed, incubated with Alexa-Fluor conjugated secondary antibodies, and mounted with ProLong Gold antifade mounting solution (Thermo Fisher Scientific). hESC-derived cardiomyocytes were stained with the mouse Anti-Human TNNT2 and rabbit Anti-Human NKX2.5 antibodies using the Human Cardiomyocyte Immunocytochemistry Kit (Thermo Fisher Scientific, A25973) according to manufacturer’s instructions.
Bright-field images were acquired using a phase contrast EVOS^TM XL Core microscope (Thermo Fisher Scientific) equipped with a 10× objective (Olympus). Immunostainings for pluripotency markers were acquired using an EVOS^TM FL Auto 2 Imaging System (Thermo Fisher Scientific) using a 1.30NA/40× oil immersion objective (Olympus). Immunostainings of hESC-derived cardiac cells were acquired using a laser scanning confocal microscope (LSM880, AxioObserver Z1, Zeiss) equipped with an Airyscan detector and a 1.4NA/60X oil objective (Zeiss), as Z-stacks of 0.3 μm steps, processed by maximum intensity projections and analyzed with ImageJ software.

Astro V., Ramirez-Calderon G., Pennucci R., Caroli J., Saera-Vila A., Cardona-Londoño K., Forastieri C., Fiacco E., Maksoud F., Alowaysi M., Sogne E., Falqui A., Gonzàlez F., Montserrat N., Battaglioli E., Mattevi A, & Adamo A. (2022). Fine-tuned KDM1A alternative splicing regulates human cardiomyogenesis through an enzymatic-independent mechanism. iScience, 25(7), 104665.

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Immunostaining of Germ Layer Differentiation

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Immunostaining of three germ layers was performed using a Human Three Germ Layer 3-Color Immunocytochemistry Kit (R&D Systems). For immunostaining of cells during cardiomyocyte differentiation, the pluripotent cells were grown and differentiated in a glass-bottomed 12-well plate (MatTek, MA), and then cells were fixed and permeated in the plate using a Human Cardiomyocyte Immunocytochemistry Kit (Thermo Fisher Scientific). The primary antibodies included mouse anti-TRA-1-60 (ESI BIO, ST11016), rabbit anti-Nanog (Stemgent, 09-0020), goat anti-Brachyury (Fisher Sciences, AF2085), rabbit anti-Pax6 (BioLegend, 901301), rabbit anti-Nkx2-5 and mouse anti-Tnnt2 (Thermo Fisher Scientific, A25973). Secondary antibodies included goat anti-mouse IgG (Alexa Fluor 488 conjugated, Thermo Fisher Scientific, A10680), donkey anti-goat IgG (DyLight 488 conjugated, Thermo Fisher Scientific, SA5-10086), goat anti-rabbit IgG (H + L) Alexa Fluor 488 (Thermo Fisher Scientific, A-11034), and donkey anti-rabbit IgG (Alexa Fluor 594 conjugated, Thermo Fisher Scientific, A21207). The images were taken using Leica DMi8 Microsystems and Zeiss LSM710 inverted confocal microscope, and then the images were processed using the Fiji software.

Liu Q., Van Bortle K., Zhang Y., Zhao M.T., Zhang J.Z., Geller B.S., Gruber J.J., Jiang C., Wu J.C, & Snyder M.P. (2018). Disruption of mesoderm formation during cardiac differentiation due to developmental exposure to 13-cis-retinoic acid. Scientific Reports, 8, 12960.

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Immunostaining of Cardiomyocyte Sarcomere Structure

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For immunostaining imaging of sarcomere structure, the differentiated cardiomyocytes were re-plated in Nunc™Lab-Tek™II glass-bottomed 8-chamber glass slides (Thermo Fisher), and then cells were fixed and permeated in the plate using a Human Cardiomyocyte Immunocytochemistry Kit (Thermo Fisher was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Scientific). The primary antibodies included: rabbit anti-cardiac troponin T (Abcam, ab45932, RRID: AB_956386) and mouse anti-α-actinin (sarcomeric) (Sigma-Aldrich, A7811, RRID: AB_476766). The secondary antibodies included: goat anti-rabbit IgG, Alexa Fluro 594 (Thermo Fisher Scientific, R37117, RRID: AB_2556545) and goat anti-mouse IgG, Alexa Fluro 488 (Thermo Fisher Scientific, A-11001, RRID: AB_2534069). The images were taken using Leica DMi8 Microsystems and Zeiss LSM710 inverted confocal microscope, and then the images were processed using the Fiji software (RRID: SCR_002285).

Liu Q., Wu H., Luo Q., Jiang C., Duren Z., Bortle K.V., Zhao M., Zhao B., Liu J., Marciano D., Lee‐McMullen B., Zhu C., Narasimha A., Gruber J.J., Lipchik A.M., Guo H., Watson N., Tsai M., Furihata T., Tian L., Wei E., Li Y., Steinmetz L.M., Wong W.H., Kay M.A., Wu J.C., & Snyder M. (2020). Tyrosine kinase inhibitors induce mitochondrial dysfunction during cardiomyocyte differentiation through alteration of GATA4-mediated networks.

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Cardiac Differentiation Phenotyping of hiPSC-CMs

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Immunostaining for cardiac troponin T (TNNT2/cTNT) and NKX2-5 was performed on day 7 after plating the hiPSC-CMs on 3DFG-MEAs by using the Human Cardiomyocyte Immunocytochemistry Kit (Life Technologies, A25973). Troponin T is a marker for cardiomyocytes, and NKX2-5 is a marker for early cardiac mesoderm. Cells were fixed with 4% (w/v) formaldehyde in DPBS for 15 min and incubated with the permeabilization solution (1% saponin in DPBS) for 15 min. Thereafter, blocking was performed using 3% bovine serum albumin for 30 min followed by incubation with primary antibodies, NKX2-5 (rabbit anti-NKX2-5 A25974) and TNNT2 (mouse anti-TNNT2 A25969), diluted 1:1000 in the blocking solution for 3 hours at room temperature. After washing three times with wash buffer, the cells were incubated for 1 hour at room temperature with secondary antibodies (Alexa Fluor 488 donkey anti-mouse and Alexa Fluor 555 donkey anti-rabbit) diluted 1:250 in blocking solution. Last, cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and imaged on an upright microscope (Nikon Eclipse FN1) with an air 20× objective or a water immersion 60× objective.

Dipalo M., Rastogi S.K., Matino L., Garg R., Bliley J., Iachetta G., Melle G., Shrestha R., Shen S., Santoro F., Feinberg A.W., Barbaglia A., Cohen-Karni T, & De Angelis F. (2021). Intracellular action potential recordings from cardiomyocytes by ultrafast pulsed laser irradiation of fuzzy graphene microelectrodes. Science Advances, 7(15), eabd5175.

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Characterizing Cardiomyocyte Differentiation

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The iPSC-derived cardiomyocytes were checked for transdifferentiation efficiency and cell protein/morphology by quantitative (flow cytometry) and qualitative (immunostaining) analyses, respectively [31 (link)]. The cells were detached with Accutase solution (Nalgene) and harvested for quantitative analysis by flow cytometry (BD FACSCanto™ II System). The fixation/permeabilization procedure was performed using the BD Cytofix/Cytoperm kit (BD Pharmingen™). The percentage of cardiomyocytes was calculated by staining with phycoerythrin- (PE-) conjugated anti-human cTnT antibody (BD Pharmingen™). All the samples were stained with the corresponding isotype control (BD Pharmingen™) to ensure specificity. Finally, the data were analyzed with flow cytometry software, and the transdifferentiation efficiency was calculated. In addition, cardiomyocytes were fixed in 4% paraformaldehyde and incubated with antibodies for immunostaining [31 (link), 33 (link)]. Antibodies against heart-associated proteins, including anti-human cTnT and NKX2.5 (Human Cardiomyocyte Immunocytochemistry Kit, Life Technologies, Invitrogen), were also used for staining to confirm the morphology of the cardiomyocytes. Finally, the nuclei were stained with DAPI. The immunofluorescence images were visualized with a microscope system, and the cell morphology was recorded at different magnifications.

Lin Y.R., Li C.J., Syu S.H., Wen C.H., Buddhakosai W., Wu H.P., Hsu Chen C., Lu H.E, & Chen W.L. (2016). Early Administration of Glutamine Protects Cardiomyocytes from Post-Cardiac Arrest Acidosis. BioMed Research International, 2016, 2106342.

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Microstructural and Cardiac Characterization of Beating EBs

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Microstructure and Cardiac Markers The microstructures of beating EBs were investigated by transmission electron microscopy (H-7600, Tokyo, Hitachi Ltd., Tokyo, Japan). According to the previously described method, 11) (link) the beating EBs were rinsed with phosphate buffer solution, fixed with 2.5% buffered glutaraldehyde, dehydrated and embedded in epoxy resin. The semi-thin sections (0.7 µm) of beating EBs were stained with 1% toluidine blue to pick up suitable cells. Ultra-thin sections (0.08 µm) of beating EBs were stained with uranyl acetate and lead citrate, and were qualitatively investigated using a transmission electron microscope.
The key markers of human cardiac lineage, NKX2-5 and TNNT2, were detected using a human cardiomyocyte immunocytochemistry kit (Life Technologies). The staining of beating EBs was performed according to an optimized staining protocol provided by the vendor. The two cardiac markers and the nuclei were stained and observed using a laser scanning microscope (LSM700, Carl Zeiss Meditec AG, Tokyo, Japan).

Aikawa N., Suzuki Y, & Takaba K. (2015). A Simple Protocol for the Myocardial Differentiation of Human iPS Cells. Biological & pharmaceutical bulletin, 38(7).

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