Total protein was extracted from tissues by RIPA lysis buffer (Beyotime), 10 μL PMSF and 10 μL phosphatase inhibitor. SDS-PAGE gel electrophoresis was utilized to separate the extracted protein. The protein was transferred to polyvinylidene fluoride membrane. After blocking with 50 g/L BAS for 1 h, the membrane was incubated with primary antibodies against HTR2B (1/3000; ab227722; Abcam, Cambridge, MA, United States), SLC5A3 (1/1000; ab113245), GAPDH (1/3000; ab8245), TNF-α (1/1000; ab215188), IL-1β (1/1000; ab200478), TGF-β (9ab208156) and IL-4 (1/1000; ab34277) at 4°C overnight. After washing the membrane with TBST, the membrane was incubated with secondary antibody (1/5000; ab7097) for 1.5 h. The ultra-sensitive ECL chemiluminescence kit was used to configure the luminescence buffer, and the band was developed.
Ab208156
Manufactured by Abcam
Sourced in United States
Ab208156 is a recombinant anti-Human Glutathione S-Transferase alpha antibody produced in E. coli. The antibody is purified using protein A affinity chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Ab66038, by Abcam (2 mentions)
Nb100 122, by Novus Biologicals (2 mentions)
Ab200478, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab113245, by Abcam (1 mentions)
Ab215188, by Abcam (1 mentions)
Ab34277, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab134175, by Abcam (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
Ab15148, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Ab18203, by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
Fluoview fv300, by Olympus (1 mentions)
Ab189494, by Abcam (1 mentions)
4 protocols using ab208156
1
Protein Extraction and Western Blot Analysis
2
Protein Quantification and Western Blot Analysis
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Protein from human tissue samples and cells was quantified by the BCA method (Thermo Fisher Scientific, Waltham, MA, USA) and separated by SDS‐PAGE. The protein was then transferred to membranes (Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk powder, the membrane was incubated with anti‐ACSS2 rabbit polyclonal antibody (1:1000, ab66038; Abcam, HongKong, China), anti‐HIF‐2α rabbit polyclonal antibody (1:1000, NB100‐122; Novus Biologicals, Littleton, Colorado, USA), anti‐transforming growth factor‐α (TGFα) rabbit mAb (1:1000, ab208156; Abcam), anti‐cyclinD1 rabbit mAb (1:5000, ab134175; Abcam), anti‐E‐cadherin rabbit polyclonal antibody (1:1000, ab15148; Abcam), anti‐N‐cadherin rabbit polyclonal antibody (1:500, ab18203; Abcam), anti‐Snail rabbit mAb (1:1000, #3876; Cell Signaling Technology, Danvers, MA, USA), anti‐vimentin rabbit mAb (1:1000, ab92547; Abcam), and anti‐β‐actin mouse mAb (1:1000, sc‐47778; Santa Cruz Biotechnology, CA, USA). After incubating overnight at 4°C, the membrane was further probed with anti‐mouse (1:4000, sc2005; Santa Cruz Biotechnology) or anti‐rabbit IgG (1:4000, sc2004; Santa Cruz Biotechnology).
3
Immunohistochemical Analysis of ACSS2, HIF-2α, and TGFα in HCC
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All HCC patient paraffin blocks used for this assay were approved by the Tianjin Medical University Cancer Institute and Hospital and were confirmed histologically by HE staining. After blocking with 3% BSA, the tissues were incubated with anti‐ACSS2 antibody (1:100, ab66038; Abcam), anti‐HIF‐2α antibody (1:250, NB100‐122; Novus Biologicals) and anti‐TGFα antibody (1:1000, ab208156; Abcam) for 30 min and then transferred to 4°C overnight. The next day, after incubating with a goat anti‐rabbit secondary antibody PV‐6000 Kit (Zhongshan Golden Bridge Biotechnology, Beijing, China) for 1 h at 37°C, the tissues were stained with DAB and hematoxylin and then dehydrated and dried before observing. Slides with the primary antibodies omitted were used as negative controls. The staining results were estimated by one researcher who was blind to the clinicopathologic staging and outcomes of all patients during observation. Five fields were selected for an average.
4
Histological and Immunofluorescent Analysis of Titanium Implant Specimens
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The undecalcified femur specimens containing titanium rods were embedded in methyl methacrylate, and cut and ground to a thickness of 40–50 μm by using Saw Microtome Leica SP1600 (Leica, Wetzlar, Germany). Histological examination of undecalcified sections were performed by stained with Von-Gieson for light microscopy according to established methods [25 (link), 26 (link)].
The titanium rods were removed and femur specimens without implant were decalcified with 10% EDTA (pH 7.4), dehydrated and embedded in paraffin prior to processing for SIRT1 and SOD2 staining. In brief, fresh bone sections were stained with individual primary antibodies to rats SIRT1 (Abcam, ab189494, 1:100) and SOD2 (Abcam, ab208156, 1:100), overnight at 4 °C. Subsequently, the secondary antibodies conjugated with fluorescence (Jackson Immuno Research, 415-605-166, 1:500; 315-605-003, 1:250) were used at room temperature for 1 h while avoiding light and observed under a confocal microscope (FLUOVIEW FV300, Olympus).
The titanium rods were removed and femur specimens without implant were decalcified with 10% EDTA (pH 7.4), dehydrated and embedded in paraffin prior to processing for SIRT1 and SOD2 staining. In brief, fresh bone sections were stained with individual primary antibodies to rats SIRT1 (Abcam, ab189494, 1:100) and SOD2 (Abcam, ab208156, 1:100), overnight at 4 °C. Subsequently, the secondary antibodies conjugated with fluorescence (Jackson Immuno Research, 415-605-166, 1:500; 315-605-003, 1:250) were used at room temperature for 1 h while avoiding light and observed under a confocal microscope (FLUOVIEW FV300, Olympus).
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