Protein concentrations of the LV tissues were determined by the BCA protein assay kit (Thermo Fisher, Erembodegem, Belgium). Western blot was performed as previously described40 (link). Briefly, equal amounts of proteins (15 µg) were separated on a 12% SDS-PAGE gel with a mini protean 3 electrophoresis system (Bio-rad Laboratories, Temse, Belgium), transferred to a polyvinylidene fluoride (PVDF) membrane and subsequently, blocked for 2 h with 5% milk in Tris-buffered solution containing 0.1% Tween-20 (TBS-T) followed by incubation overnight at 4 °C in the presence of a specific endothelin-1 antibody (1/2500, Abcam, ab117757, Cambridge, United Kingdom), NOX2 antibody (1/2500, Abcam, ab31092, Cambridge, United Kingdom) or an OXPHOS antibody (1/1000, Abcam, ab110411, Cambridge, United Kingdom). Horseradish peroxidase-conjugated secondary antibodies (DAKO, Belgium) at a dilution of 1/2000 were used. Both primary and secondary antibodies were diluted in 5% milk-TBS-T. Visualization was performed with the enhanced chemiluminescence (ECL) technique using the Pierce ECL Plus western Blotting Substrate (Thermo Fisher, Erembodegem, Belgium). Data were normalized to β-actin protein levels.
Ab117757
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab117757 is a primary antibody that recognizes the Human PD-L1 protein. It is suitable for use in various immunological applications such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Ab7817, by Abcam (2 mentions)
Ab31092, by Abcam (1 mentions)
Ab110411, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Mini protean 3 electrophoresis system, by Bio-Rad (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Dako envision system, by Agilent Technologies (1 mentions)
Ab76198, by Abcam (1 mentions)
Dako envision dual link system hrp dab, by Agilent Technologies (1 mentions)
Entellan, by Merck Group (1 mentions)
Ab85163, by Abcam (1 mentions)
G2026, by Wuhan Servicebio Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab34710 100, by Abcam (1 mentions)
Glycergel, by Agilent Technologies (1 mentions)
Ab183495, by Abcam (1 mentions)
Ab174279, by Abcam (1 mentions)
D110001, by Sangon (1 mentions)
7 protocols using ab117757
1
Western Blot Analysis of Endothelin-1, NOX2, and OXPHOS
2
Muscle Tissue Immunohistochemistry for ET-1 and eNOS
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Immunohistochemically analysis for endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS) was performed with the use of a mouse polyclonal antibody against ET-1 (Cat. No. ab117757; Abcam Ltd, Cambridge, UK) and eNOS (Cat. No. ab76198; Abcam Ltd). Cross sections of the thigh and calf muscle tissue were embedded in paraffin and cut in slices 5-μm thick. The sections were deparaffinized with xylene and rehydrated with graded ethanol. The activities of endogenous peroxidase and alkaline phosphatase were blocked by boiling for 20 minutes using 0.01 M citrate buffer (pH 6.0). CAS-BLOCK (Cat. No. 008120; Invitrogen Corporation, Camarillo, CA, USA) were added to inhibit nonspecific protein binding. The primary antibodies were diluted (1:200) and incubated with the tissue sections for 3 hours at room temperature. The sections were then washed three times with phosphate-buffered saline and incubated with a biotin-labeled secondary antibody was applied for 30 minutes, followed by DAKO EnVision system (Cat. No. K5007; Santa Clara, CA, USA) for 30 minutes at room temperature. They were then counterstained with Mayer's hematoxylin stain, dehydrated, and cover slipped.
3
Immunohistochemical Analysis of ET-1 in CCA
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The expression of inflammatory factors, Endothelin-1 (ET-1), in CCA were investigated at 7 days after LED-induced carotid thrombosis. Sections were processed using the cross sections of CCA (10 μm thickness) and stored in OCT at − 80 °C before use. Sections were handled to remove OCT with phosphate-buffered saline (PBS) for 10 min. Next, Dako EnVision®+ Dual Link System-HRP (DAB+) (CAT#K4065, Agilent Technologies, CA, USA) was used according to manufacturer’s instruction. Rabbit polyclonal antibody against ET-1 (1:200; ab117757, Abcam, Cambridge, MA, USA) was applied for 30 min, and labelled polymer (CAT#K4003, Agilent Technologies, CA, USA) was used to cover specimen for another 30 min. The specimens were then covered with substrate-chromogen solution for 5 min and after mounted with a mounting medium, entellan (CAT#107961, Merck, Germany), the specimens were cover-slipped for microscopic study.
4
Quantitative Analysis of Endothelin Pathway
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Lung tissues were homogenized and centrifuged at 12000 rpm for 10 min. Supernatants were extracted and the protein concentration was determined with a BCA kit (G2026; Servicebio). Protein solution was added into the loading buffer (G2013; Servicebio) in a ratio of 4:1 and denatured in boiling water for 15 min. Samples were loaded on 5% SDS-PAGE gels (Servicebio), then separated by electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA, United States). After being blocked in 5% fat-free milk for 1 h, membranes were incubated overnight with the following primary antibodies: ET-1 (ab117757; Abcam, Cambridge, United Kingdom), ET-A (ab85163; Abcam), ET-B (ab262694; Abcam), ECE-1 (sc-376017; Santa Cruz Biotechnology, Dallas, TX, United States), and GAPDH (ab9485; Abcam). The next day, the membranes were incubated in horseradish peroxidase-conjugated secondary antibodies, which were diluted 3000 times with Tris-buffered saline Tween-20. Bands were visualized with ECL solution (Servicebio) and the band intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MA, United States).
5
Immunohistochemical Analysis of Kidney
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For immunoreactivity 5-μm sections of the kidneys fixed in 3% PFA were blocked with 10% horse serum/1% BSA in PBS and were incubated either with rabbit anti ET-1 (ab117757, Abcam, Cambridge, UK), mouse anti-αSMA (ab7817, Abcam, Cambridge, UK), or rabbit anti-col1a1 (ab34710-100, Abcam) in different experimental approaches at 4°C overnight. After washing with BSA/PBS, sections were incubated with Cy3 and Cy5 secondary antibodies (Dianova, Hamburg, Germany), mounted with Glycergel (Agilent, Waldbronn, Germany), and viewed with an Axio Observer.Z1 Microscope.
6
Protein Expression Analysis of HUVECs
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Protein lysates of HUVECs were prepared using Radio Immuno Precipitation Assay (RIPA) Lysis Buffer. Western blot analysis was performed using antibodies against FURIN (Abcam; ab183495), endothelin‐1 (Abcam; ab117757), vascular cell adhesion molecule‐1 (VCAM1) (Abcam; ab174279), MCP1 (monocyte chemotactic protein‐1) (Abcam; ab151538), and beta‐Actin (Sangon Biotech; D110001).
7
Endothelin-1 and TGF-β1 Signaling Pathways
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Antibodies used were α-smooth muscle actin (α-SMA) (ab7817; Abcam, Cambridge, UK), pSmad3 (ab51451; Abcam), Smad3 (ab40854; Abcam), VEGF (ab1316; Abcam), cleaved caspase-3 (#9661; Cell Signaling Technology, Danvers, MA, USA), ET-1 (ab117757; Abcam), CD31 (sc-1506; Santa Cruz Biotechnology, Santa Cruz, CA, USA), ETRA (ab117521; Abcam), ETRB (ab117529; Abcam), GAPDH (#5174; Cell Signaling Technology), anti-rabbit HRP-linked IgG (#7074; Cell Signalling Technology) and anti-mouse IgG HRP-linked antibody (#7076; Cell Signaling Technology). For fluorescence microscopy, we used goat or donkey secondary antibody conjugated with Alexa Fluor-488 and Alexa Fluor-555 (Abcam). Human rET-1 (100-21; PerproTech, London, UK) and human rTGF-β1 (240-B; R&D Systems, Minneapolis, MN, USA) were used in vitro to treat cells.
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