Myocardial tissues and cells were collected and lysed using ultrasonic cell disruption on ice in radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) for 30 min. The samples were centrifuged at 12,000 × g for 30 min at 4 °C to collect the supernatant. Protein concentrations were determined using a BCA protein assay kit (Sigma-Aldrich Chemical Company, St Louis, MO, USA). Protein lysates (30 μg) were loaded onto 12% sodium dodecyl sulfate–polyacrylamide gels for electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). One hour of blocking was done at room temperature with 5% BSA (Gibco, Carlsbad, CA, USA). Membranes were incubated at 4 °C overnight with primary antibodies as follows: G9a (1:1000, ab185050, Abcam, Cambridge, UK), BDNF (1:2000, GTX132621, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), H3K9me2 (1:1000, GTX54102, GeneTex), α-SMA (1:2000, GTX100034, GeneTex), FN1 (1:1000, ab268020, Abcam), p-TrkB (1:1000, ab229908, Abcam), TrkB (1:1000, GTX54857, GeneTex), GAPDH (1:10,000, ab181602, Abcam). After a wash, blots were incubated with goat anti-rabbit IgG-horseradish peroxidase (1:5000, ab6721, Abcam) for 60 min at room temperature. The blot was visualized with an ECL kit (Beyotime) and quantified with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
Gtx100034
Manufactured by GeneTex
Sourced in United States
The GTX100034 is a laboratory centrifuge designed for general-purpose applications. It is capable of separating biological samples, such as cells or proteins, based on differences in their densities and sedimentation rates. The centrifuge features adjustable speed and time settings to accommodate various sample volumes and separation requirements.
Automatically generated - may contain errors
Lab products found in correlation
Ab229908, by Abcam (1 mentions)
Ab268020, by Abcam (1 mentions)
Ab185050, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab181602, by Abcam (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
Ab6721, by Abcam (1 mentions)
Gtx54102, by GeneTex (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Ab8295, by Abcam (1 mentions)
F3648, by Merck Group (1 mentions)
Sc 47724, by Santa Cruz Biotechnology (1 mentions)
G8795, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ab6640, by Abcam (1 mentions)
Picrosirius red stain kit, by Polysciences (1 mentions)
3 30 diaminobenzidine, by Agilent Technologies (1 mentions)
Nbp1 85047, by Novus Biologicals (1 mentions)
6 protocols using gtx100034
1
Proteomic Analysis of Myocardial Tissues
2
Immunofluorescence Staining of Cell Markers
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Cells were fixed with 4% paraformaldehyde for 30 min. Following this, the cells were incubated with serum for 30 min to block non-specific binding sites. The primary antibody was added and incubated overnight after removal of the serum. The secondary antibody was then introduced and allowed to interact with the cells for 1 h, shielded from light. DAPI was subsequently added to stain the cell nuclei. The specimens were then observed using confocal scanning laser microscopy (Nikon A1R; Nikon Corporation). Integrated fluorescence intensity was quantified using ImageJ software (National Institute of Health, Bethesda, MD, USA). The antibodies employed in this study recognized KRT19 (1:300, 60187-1-lg, Proteintech), MIF (1:300, 20415-1-AP, Proteintech), CD3 (1:100, 17617-1-AP, Proteintech), CD74 (1:500, GTX110477, Proteintech), C-X-C motif chemokine receptor 4 (1:100, 60042-1-Ig, Proteintech) and α-SMA (1:500, GTX100034, GeneTex).
3
Immunoblotting Reagents and Protocols
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The antibody against fibronectin (FN, F3648) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, G8795) were purchased from Sigma-Aldrich and Santa Cruz Biotechnology (sc-47724, Dallas, TX, USA). The antibody for cardiac troponin-T (TnT, ab8295) was acquired from Abcam (Cambridge, MA, USA). The antibodies against alpha-smooth muscle actin (αSMA) were purchased from Abcam (ab49481) and GeneTex (GTX100034, San Antonio, TX, USA). All antibodies were used according to the manufacture’s recommendations. The HRP-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
4
Histological and Immunohistochemical Evaluation of Liver and Intestinal Tissues
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The formalin-fixed liver sections were stained with H & E, Sirius red, and Masson’s trichrome to detect any liver injury and fibrosis. Further, intestine sections were stained with H & E. The liver HE-stained sections were scored using the NAFLD activity score (NAS) system (Brunt et al., 2011 (link)) by our co-author H.-S.L.—an experienced pathologist—and the degree of fibrosis was evaluated by Sirius red stain (Picrosirius Red Stain Kit, Polysciences, Inc., Warrington, PA, United States) and Masson’s trichrome staining (Muto pure chemical co., Ltd.). NIH ImageJ was used to estimate the length and width of gut villi and crypts.
Paraffin-embedded liver sections were stained for F4/80 (ab6640, abcam®) and stained for alpha-smooth actin (a-SMA) (GTX100034, GeneTex). Paraffin-embedded intestine sections were stained for ZO-1 (NBP1-85047, Novus). Standard immunohistochemical (IHC) staining procedures were employed; liver and intestine sections were incubated with a specific primary antibody, as described earlier, followed by incubation with an HRP-linked secondary antibody (Dako, Glostrup, and Denmark) and 3,30-diaminobenzidine (Dako, Glostrup, and Denmark) and were scanned using the NanoZoomer Digital Pathology system.
Paraffin-embedded liver sections were stained for F4/80 (ab6640, abcam®) and stained for alpha-smooth actin (a-SMA) (GTX100034, GeneTex). Paraffin-embedded intestine sections were stained for ZO-1 (NBP1-85047, Novus). Standard immunohistochemical (IHC) staining procedures were employed; liver and intestine sections were incubated with a specific primary antibody, as described earlier, followed by incubation with an HRP-linked secondary antibody (Dako, Glostrup, and Denmark) and 3,30-diaminobenzidine (Dako, Glostrup, and Denmark) and were scanned using the NanoZoomer Digital Pathology system.
5
Immunohistochemical Analysis of α-SMA in Myocardial Tissues
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Myocardial tissues were routinely paraffin-embedded and sectioned at 4 μm. The sections were first dewaxed and rehydrated. After blockage of endogenous peroxidase with 3% hydrogen peroxide solution, the sections were blocked with 100 μL of 5% BSA at 37 °C for 0.5 h. The sections were incubated overnight in 50 μL antibody against α-SMA (1:500, GTX100034, GeneTex) at 4 °C and with biotin-labeled secondary antibody goat anti-rabbit working solution (1:2000, ab205718, Abcam) at 37 °C for 0.5 h. Then, the sections were incubated with streptavidin-peroxidase solution (Solarbio, Beijing, China) for 0.5 h at 37 °C. Finally, the sections were treated with diaminobenzidine, counter-stained with hematoxylin for 30 s, dehydrated, fixed, observed and counted under a microscopy (Olympus Optical Co., Ltd., Tokyo, Japan).
6
Protein Extraction and Western Blot Analysis
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The extraction of total protein was performed by lysing the cultured cells using radioimmunoprecipitation assay lysis buffer (RIPA; Beyotime, Shanghai, China) supplemented with phenylmethylsulfonylfluoride (PMSF; Beyotime, Shanghai, China). The concentrations of proteins were tested by bicinchoninic acid protein assay kit (BCA; Beyotime, Shanghai, China). Equivalent proteins were isolated by means of 12% SDS-PAGE (Beyotime, Shanghai; P0012A) and then were transferred onto a polyvinylidene fluoride (PVDF; Beyotime, Shanghai; FFP28) membrane. The immunoassay of proteins was performed via specific antibodies. The chemiluminescence detection was conducted by means of ECL (Amersham Pharmacia, Piscataway, NJ, USA) system, and density analysis was performed by ImageJ software. Primary antibodies were as follows: SOX18 (ab23342, 1: 500, ABCAm, USA), E-cadherin (ab15148, 1: 1000, ABCAm, USA), vimentin (ab137321, 1: 500, ABCAm, USA), AMPK (ab32047, 1: 1000, ABCAm, USA), p-AMPK (ab92701, 1: 1000, ABCAm, USA), mTOR (ab2732, 1: 2000, ABCAm, USA), p-mTOR (ab109268, 1: 1000, ABCAm, USA), and α-SMA (GTX100034, 1: 1000, GeneTex, USA). The antibodies against GAPDH served as an internal reference. HRP-conjugated goat anti-rabbit IgG (ab6741, 1: 5000, ABCAm, USA) were used as secondary antibodies.
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