Cells were fixed in 4% paraformaldehyde (Millipore-Sigma, St. Louis, MO) for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature for nuclear markers. Cell fixation and permeabilization for cytoskeletal markers was done with cold (−20°C) methanol for 3–5 min. Samples were washed three times (5 min each time) with PBS between each step and blocked with 3% normal donkey serum (NDS; Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS for 30 min. Samples were incubated at 4°C with primary antibodies for: cardiac troponin T (TNNT2; rabbit; ab45932; Abcam, Cambridge, MA), α-actinin (ACTN1; mouse; sc-15335) and GATA4 (rabbit; sc-9053; both from Santa Cruz Biotechnology, Dallas, TX). Incubation with secondary antibodies was performed at room temperature for 1 h with donkey anti-rabbit or anti-mouse antibodies conjugated to DyLight 488 or 549 (Jackson ImmunoResearch Inc., West Grove, PA). Nuclear DNA was stained with DAPI (Sigma-Aldrich, St. Louis, MO). Controls were stained with IgG instead of with a primary antibody. Immunostaining was visualized with a Leica TCS SPE confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL).
Sc 9053
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-9053 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed for scientific research purposes. The core function of Sc-9053 is to provide a tool for researchers to conduct their experiments and studies. Further details on the specific features and intended use of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Tcs spe confocal microscope, by Leica (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab45932, by Abcam (1 mentions)
α actinin, by Santa Cruz Biotechnology (1 mentions)
Sc 15335, by Santa Cruz Biotechnology (1 mentions)
Dylight 488 or 549, by Jackson ImmunoResearch (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
Sc 6216, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Ab236101, by Abcam (1 mentions)
Ab5245, by Abcam (1 mentions)
Pierce ecl system, by Thermo Fisher Scientific (1 mentions)
Nb100 2219, by Novus Biologicals (1 mentions)
Nb100 479, by Novus Biologicals (1 mentions)
3 protocols using sc 9053
1
Cardiac Cell Phenotyping by Immunostaining
2
Quantification of Cardiac Transcription Factor GATA4
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The basal transversal mid‐section of the LV was dissected and snap frozen in liquid nitrogen. Next, the tissues were processed for total and nuclear protein extractions, as previously described.30 , 33 Equal amounts of protein were loaded onto 10% SDS‐PAGE and transferred to nitrocellulose membranes. Primary antibodies against total GATA4 (sc‐9053, Santa Cruz), phosphorylated GATA4 at S105 (44‐948, Invitrogen, CA, USA) and Lamin B (sc‐6216, Santa Cruz) were used according to the manufacturers’ instructions and detected with fluorescent secondary antibodies: anti‐rabbit and antimouse IgG Alexa Fluor 680 (A21076 and A21058, Invitrogen), anti‐rabbit IgG IRDye 800 (926‐32211, LI‐COR Biosciences, Lincoln, NE, USA) and anti‐goat IRDye 700 (605‐730‐125, Rockland). Protein levels were visualized with Odyssey Fc imaging system (LI‐COR Biosciences).
3
Cardiac Protein Detection and Quantification
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For GATA4, pGATA4 and BNP thirty μg and for MEF2C hundred μg of total protein from heart tissue was lysed in EBC + urea buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 3 M urea) resolved by SDS-PAGE, transferred to PVDF membrane, blotted, and probed with the following primary antibodies: anti-GATA4 pSer105 (1:600 ab5245, Abcam), anti-GATA4 (1:1000 sc-9053, Santa Cruz Biotechnology), anti-BNP (1:500 ab236101, Abcam), anti-vinculin (1:200 V4505, Merck) and anti-MEF2C (1:7500 sc-313×, Santa Cruz Biotechnology) followed by a HRP-conjugated secondary antibody (1:5000; Bio-Rad). For blotting of HIF1α and HIF-P4H-2 hundred ug of total protein from heart tissue lysed using EBC + urea lysis buffer and was resolved by SDS-PAGE, transferred with nitrocellulose membrane, blotted and probed with anti-HIF1α (1:500, NB100-479 Novus Biologicals) and anti-HIF-P4H-2 (1:500, Novus Biologicals NB100-2219)primary antibodies followed by HRP-conjugated secondary antibody. The Pierce ECL system (Ther-moScientific) was used for detection. Western blot images were quantified with fiji (ImageJ) software. For original blots see (Supplementary Fig. S1-S8).
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