Western blot assays were performed as previously described34 (link). Briefly, the total cellular proteins were extracted by using reagents in a Radio Immunoprecipitation Assay kit (Beyotime, Shanghai, China). The extracted proteins were then separated by SDS-PAGE, and the individual protein bands were transferred onto a PVDF membrane, which was subsequently blocked with 5% skim milk. The membrane was then incubated with primary antibodies against E-cadherin (1:800, ab76055, Abcam, Cambridge, MA, USA), FN (1:5000, ab45688, Abcam, USA), β-catenin (1:8000, ab32572, Abcam, USA), α-SMA (1:1000, ab28052, Abcam, USA), LC3B (1 μg/mL, ab48394, Abcam, USA), p62 (1:2000, ab155686, Abcam, USA), or the internal reference GAPDH (1:20000, ab128915, Abcam, USA). Next the membrane was incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies. An enhanced chemiluminescence detection system was used to detect areas of luminescence, and the relative-staining intensity of each protein band was quantitated using Image Plus Pro software (Ver. 6.0, Media Cybernetics, Inc., Rockville, MD, USA).
Ab28052
Manufactured by Abcam
Sourced in United States
Ab28052 is a primary antibody that recognizes the CD3 epsilon subunit. It is a mouse monoclonal antibody that is designed for use in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab62623, by Abcam (2 mentions)
Ab64715, by Abcam (2 mentions)
Vectastain abc hrp kit, by Vector Laboratories (2 mentions)
Ab9722, by Abcam (2 mentions)
Ab2728, by Abcam (2 mentions)
Ab45688, by Abcam (1 mentions)
Ab76055, by Abcam (1 mentions)
Ab155686, by Abcam (1 mentions)
Radioimmunoprecipitation assay kit, by Beyotime (1 mentions)
Ab48394, by Abcam (1 mentions)
Ab128915, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Il 11rα1, by Santa Cruz Biotechnology (1 mentions)
Interleukin 6 (il 6), by Santa Cruz Biotechnology (1 mentions)
Ab211542, by Abcam (1 mentions)
Ab87312, by Abcam (1 mentions)
Alexa fluor 488 donkey anti goat igg h l, by Thermo Fisher Scientific (1 mentions)
Cm1850, by Leica (1 mentions)
Ab2864, by Abcam (1 mentions)
Ab2861, by Abcam (1 mentions)
10 protocols using ab28052
1
Western Blot Analysis of EMT Markers
2
Immunohistochemical Analysis of Cellular Markers
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Immunohistochemical staining was performed as previously described. Primary antibodies against p16 (ab211542; Abcam), p53 (sc‐126; Santa Cruz Biotechnology), acetyl‐histone H3 (Lys9/Lys14; #9677; Cell Signaling Technology), 8‐OHdG (ab62623; Abcam), IL‐1β (ab9722; Abcam), CD3e (sc‐20,047; Santa Cruz Biotechnology), IL‐6 (sc‐1265; Santa Cruz Biotechnology), TNF‐α (sc‐52,746; Santa Cruz Biotechnology), α‐SMA (ab28052; Abcam), Collagen 1 (#1310‐08; Southern Biotech), TGF‐β1 (ab64715; Abcam), and IL‐11 (sc‐133,063; Santa Cruz Biotechnology), and IL‐11Rα1 (sc‐130,920; Santa Cruz Biotechnology). After washing, the sections were incubated with secondary antibody (biotinylated IgG; Sigma‐Aldrich), washed, and processed using Vectastain ABC‐HRP kits (Vector Laboratories).
3
Immunofluorescent Imaging of Cardiac Cryosections
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Heart cryostat sections (20 μm thick) were cut at –20°C (Leica CM1850, Leica, Germany) and mounted directly on slides, then dried and fixed with 4% PFA (30 min), permeabilised with 0.25% Triton X-100, and blocked with 10% donkey serum +1% BSA. The following primary antibodies were applied overnight: OTR (Abcam, ab87312, 1 : 200), a skeletal muscle actin (a-SMA) (Abcam, ab28052, 1 : 100). After washing with PBS, appropriate secondary antibodies (Donkey anti-Mouse IgM (H + L) Alexa Fluor® 594 or Donkey anti-Goat IgG (H + L) Alexa Fluor® 488, 1 : 500, ThermoFisher Scientific) were applied for 2 h at room temperature (RT). Cell nuclei were contra-stained (20 min/RT) with Hoechst 33258. Images were obtained using a Zeiss Confocal Laser Microscope LSM 710 (Zeiss LSM 710, Carl Zeiss, Oberkochen, Germany) with a coherent multiphoton unit.
4
Cardiac Protein Expression Analysis
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Crude tissue homogenate from cardiac ventricles was separated by electrophoresis and transferred onto PVDF membranes. Membranes were incubated with primary antibodies: anti‐PAD2 (1:1000, Abcam, ab16478), anti‐RyR2 (1:5000, Abcam, ab2728), anti‐phosphorylated‐S2808‐RyR (1:5000, donated by Andrew R Marks, Columbia University), anti‐dihydropyridine receptor (1:1000, Abcam, ab2864), anti‐calsequestrin 2 (1:1000, Abcam, ab3516), anti‐SR Ca2+‐ATPase 2a (SERCA 2a; 1:1000, Abcam ab2861), anti‐phospholamban (1:1000, AbCam, ab2865), α‐actin (1:1000, Abcam ab28052), anti‐citrulline antibody (1:1000, Millipore, 07–377), Malondialdehyde (MDA) antibody (1: 1000, Abcam, ab27642), GAPDH (1:1000, Abcam, ab9485), and infrared‐labeled secondary antibodies (1:5000, Licor IRDye 680 and IRDye 800). Immunoreactive bands were analyzed using the Odyssey Infrared Imaging System. Band densities were quantified with Image J and normalized to GAPDH. All the experiments were conducted blinded regarding the treatment of the mice.
5
Immunoprecipitation of Cardiac Proteins
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Dynabeads Protein G (Invitrogen) were incubated with anti‐α‐actin (Abcam, ab28052) or RyR2 antibody (Abcam, ab2728) for 40 min at RT to allow the cross‐binding between beads and antibody, then the beads were washed three times with phosphate buffer solution (PBS). Cardiac ventricle lysate (500 μg) was incubated at 4°C overnight with the beads conjugated to the anti‐α‐actin antibody. The immunocomplex was washed once with lysis buffer and then three times with PBS. Finally, the immunoprecipitate was collected in 50 μl of Laemmli buffer 2× (Bio‐Rad #161–0737, with β‐Mercaptoethanol 10% and DTT 0.05 M). Proteins were separated by electrophoresis and immunoblots were performed as described above and quantified relative to the RyR2 or α‐actin expression.
6
Glucose Metabolism Pathway Analysis
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Cell culture media (Dulbecco’s modified Eagle’s medium, phosphate-buffered saline and penicillin-streptomycin), sodium pyruvate, sodium palmitate and 2-deoxy-D-glucose were purchased from Sigma Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), horse serum, cell extraction buffer and cocktail inhibitor were acquired from Life Technologies (Inchinnan, UK). Tritiated glucose, D-[3-3H], was obtained from Perkin Elmer (Boston, MA, USA). Mitochondrial ATP monitoring reagent was acquired from Biothema (Handen, Sweden). Protein assay reagents were purchased from BioRad (Copenhagen, Denmark). Anti-PDK4 (ab214938), anti-PPAR alpha (ab24509), anti-PPAR delta (ab23673), goat anti-Mouse (IRDye® 800CW) (ab216772), goat Anti-Rabbit (IRDye® 680RD) (ab216777) and anti-skeletal muscle alpha-actin (ab28052) antibodies were acquired from Abcam (Cambridge, UK). Anti-Phospho-FoxO1 (Ser256) (#9461) and Anti-FoxO1 (#2880) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
7
Immunofluorescent Analysis of Cell Markers
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Primary antibodies against IL-11 (sc-133063, Santa Cruz Biotechnology Inc.), vimentin (#5741, Cell Signaling Technology), p53 (#2524, Cell Signaling Technology), TGF-β RII (sc-17792, Santa Cruz Biotechnology Inc.), IL-11Rα1 (sc-130920, Santa Cruz Biotechnology Inc.), α-SMA (ab28052, Abcam), SFTPC (ab211326, Abcam), p16 (#MA5-17142, Invitrogen Inc.), ERK1/2 (#4695, Cell Signaling Technology), and pERK1/2(Thr202/Tyr204) (#4370, Cell Signaling Technology) and affinity-purified Alexa Fluor 488-conjugated and 594-conjugated secondary antibodies (Life Technologies Corporation, USA) were used. Details are provided in Supporting Information 3.
8
Immunohistochemical Staining of Biomarkers
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Staining was performed as previously described4 (link),5 (link),23 (link). Primary antibodies against p53 (#2524, Cell Signaling Technology, Beverly, MA, USA), 8-OHdG (ab62623, Abcam, Cambridge, MA, USA), IL-1β (ab9722, Abcam), IL-6 (sc-1265, Santa Cruz Biotechnology Inc., Dallas, TX, USA), TNF-α (sc-52746, Santa Cruz Biotechnology Inc.), α-SMA (ab28052, Abcam), collagen 1 (#1310-08, Southern Biotech, Birmingham, AL, USA), fibronectin (#SAB4500974, Sigma-Aldrich), TGF-β1 (ab64715, Abcam), and IL-11 (sc-133063, Santa Cruz Biotechnology Inc.). After washing, the sections were incubated with secondary antibody (biotinylated IgG; Sigma-Aldrich), washed and processed using Vectastain ABC-HRP kits (Vector Laboratories Inc., Burlingame, CA, USA).
9
Multicolor Immunofluorescence Staining for Cardiac and Skeletal Muscle
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IF counterstaining was performed according to standard procedures. Briefly, paraffin tissue sections were deparaffinized in xylene, and rehydrated in graded ethanol before antigen retrieval in citrate buffer (10 mM, at 98° C) for 20 min. After cooling down, the sections were washed in PBS with 0.3% Triton X-100 and blocked with 5% donkey normal serum for 1 h at room temperature. Then, the sections were incubated overnight with rabbit anti-cardiac troponin T (ab209813, Abcam) and mouse anti-α skeletal muscle actin (α-SMA) primary antibodies (ab28052, Abcam). After washing with PBS, the sections were stained with goat anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150077, Abcam) and goat anti-mouse IgG H&L secondary antibodies (Alexa Fluor® 488) (ab150113, Abcam); for PTX3-Immunofluorescence staining, the PTX3 ((ab125007, 1:50 dilution) and goat anti-rabbit IgG H&L (Alexa Fluor® 488) were used. Then the sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, blue) to stain nuclei. Images were captured under a confocal microscope.
10
Quantifying Endothelin Receptor Expression
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Three sections from each graft were stained with HE and the thickness was measured. Immunohistochemistry and immunofluorescence was performed as described previously [16] . The sections were incubated overnight with anti-ET A R (Abcam, ab85163) and anti-ET B R (Abcam, ab117529) for immunohistochemistry. To evaluate the expression of ET B R in endothelial and smooth muscle cells, the sections were double stained by anti-CD 31(Abcam, ab119339) and anti-α-SMA (Abcam, ab28052) with anti-ET B R (Abcam, ab117529) by immunofluorescence. Negative controls were included using isotypematched control antibodies (Abcam). In the control experiments, either the primary antibody or secondary antibody was omitted. The stained arterial segments were observed under a confocal microscope (Nikon, C1plus, Nikon Instruments, Inc, NY, USA) and analyzed by IMAGE-PRO PLUS. For quantification, the mean optical densities of the ET A R and ET B R staining in the immunohistochemistry images were determined using IMAGE-PRO PLUS. In each section, the optical density was measured at 4 preset areas, and the mean optical density was obtained from 6 vessels.
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