Undifferentiated cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100, blocked in 3% horse serum and stained for Nanog (R&D Systems, Abingdon, UK), Oct3/4 and Tra-1-60 (both from SantaCruz Biotechnology, Dallas, TX, USA). EBs differentiated to the three germ layers were fixed in cold methanol, permeabilized and blocked with 10% goat serum and stained for α-fetoprotein, β-tubulin III (both from Sigma-Aldrich, St. Louis, MO, USA) and Muscle actin (Dako, Glostrup, Denmark). Disaggregated beating bodies were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked in 3% goat serum and stained for cardiac troponin T, cardiac troponin I, and alpha actinin (all from Abcam, Cambridge, UK). Appropriate secondary antibodies were used and cells were counterstained and mounted with ProLong Gold Antifade Reagent with DAPI.
Alpha actinin
Manufactured by Abcam
Sourced in United Kingdom
Alpha actinin is a cytoskeletal protein that crosslinks actin filaments. It plays a role in the organization and stability of the actin cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Tra 1 60, by Santa Cruz Biotechnology (1 mentions)
Cardiac troponin t, by Abcam (1 mentions)
Oct3 4, by Santa Cruz Biotechnology (1 mentions)
Nanog, by R&D Systems (1 mentions)
α fetoprotein, by Merck Group (1 mentions)
Cardiac troponin 1, by Abcam (1 mentions)
β tubulin 3, by Merck Group (1 mentions)
Gpr91, by Novus Biologicals (1 mentions)
Connexin 43, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568, by Abcam (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Normal goat serum (ngs), by Abcam (1 mentions)
Alexa fluor 647 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Beta 3 tubulin, by Abcam (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Black bottom 96 well plate, by Corning (1 mentions)
Anti mouse alexa fluor 488, by Abcam (1 mentions)
5 protocols using alpha actinin
1
Immunostaining Undifferentiated and Differentiated Cells
2
Histopathological Evaluation of Cardiomyocytes
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The hearts and lungs were excised, washed with saline solution and placed in 10% formalin. Several heart sections (4–5 μm thick) were prepared and stained with hematoxylin and eosin (HE) or with Masson for histopathology and then visualized by light microscopy. The evaluation of the cross-sectional area of the cardiomyocytes was outlined previously [24 (link)]. Immunofluorescence staining was performed using primary antibodies to detect GPR91 (1:50; Novus, USA) and alpha actinin (1:50; Abcam, USA), followed by incubation with a fluorescein isothiocyanate-conjugated secondary antibody (1:100; Bioworld, China). For the negative control experiments, the primary antibodies were omitted.
3
Immunofluorescence Characterization of hiPSC-CMs
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Human iPSC-CMs were fixed in 4% paraformaldehyde for 20 min, followed by washing (PBS) wash. Nonspecific antigenic sites of the fixed cells were blocked with 5% FBS (FBS; Gibco, Gaithersburg, MD, USA) for 1 h at 4 °C. Primary antibodies, including alpha-actinin (Abcam; catalog no. ab68194, Cambridge, UK) and connexin 43 (Thermo Fisher Scientific; catalog no. 71-0700) were hybridized at 4 °C overnight. The samples were then rinsed with PBS and incubated with secondary antibodies: goat anti-mouse IgG (H&L), Alexa Fluor™ 488 (Abcam; catalog no. ab175473), and goat anti-rabbit IgG (H&L), Alexa Fluor 568 (Abcam; catalog no. ab175471), at room temperature for 1 h. All primary and secondary antibodies were prepared in PBS containing 2% BSA (Thermo Fisher Scientific, catalog no. 15260037). Fluorescence images were captured using an inverted fluorescence microscope (OLYMPUS; catalog no. BX51; Tokyo, Japan). The acquired fluorescent images were processed and quantified using ImageJ software.
4
Immunostaining of Neuronal and Glial Markers in Biobots
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Tissues were rinsed with PBS and fixed in 4% (vol/vol) paraformaldehyde. Prior to immunostaining, tissues were permeabilized with 0.3% (vol/vol) Triton X-100 (EMD Millipore) and blocked with 3% Normal Goat Serum (NGS, Abcam, Cambridge, UK) for 30 min. Tissues were incubated with alpha-actinin (1:2000, Abcam), Beta-III Tubulin (1:2000, Abcam), conjugated alpha bungarotoxin (1:1000 EMD Millipore), or glial fibrillary acidic protein (1:20 000, Abcam) antibodies for 48 h at 4 °C and washed with PBS. Biobots were incubated with Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 568 goat anti-chicken IgG, and Alexa Fluor 647 goat anti-rabbit IgG secondary antibodies (1:1000, Invitrogen, Waltham, MA) in PBS for 2 h at room temperature in the dark.
5
Immunocytochemistry of hiPSC-Derived Cardiomyocytes
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For immunostaining, hiPSC-CMs were plated on thin black bottom 96 well plates (Corning). Cells were washed using PBS followed by fixing using 5% PFA (Sigma-Aldrich) for 15 min followed by 2x PBS washes. Fixed cells were then washed for 15 min in 0.1% Triton X-100 (Sigma-Aldrich, Gillingham, UK) in PBS followed by 3x further washes in PBS. Fixed cells were then blocked in 4% Goat Serum (Sigma-Aldrich, Gillingham, UK) in PBS for 1 h followed by a further 5 min PBS wash. Primary antibody was added to the plates, diluted in 4% Goat Serum in PBS (Alpha Actinin 1:800, Abcam) and left overnight at 4 °C on a rocking plate. The following day fixed cells were washed with 0.1% Tween (Sigma-Aldrich, Gillingham, UK) in PBS (3 × 10 min) followed by secondary antibody staining [Anti-Mouse Alexa Fluor® 488 (Abcam, Cambridge, UK) diluted 1:500 in 4% Goat Serum (Sigma-Aldrich, Gillingham, UK) in PBS] for 1 h at room temperature. The fixed cells were then washed 3 times with 0.1% Tween-PBS (Sigma-Aldrich, Gillingham, UK) followed by incubation in PBS supplemented with DAPI (Tocris, Abingdon, UK) at 1:500 for 10 min. A further wash was performed using PBS after which the fixed cells were submerged in PBS and the plate wrapped in foil prior to imaging using the EVOS M5000 imaging system.
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