The Huh7 and HCCLM3 cells were trypsinized and seeded in 24-well plates. After being incubated at 37 ℃ with 5% CO2 for 48 h, the cells were fixed with 4% paraformaldehyde for 20 min at RT, and their endogenous peroxidase was blocked with 3% H2O2 for 15 min. 0.5% Triton X-100 was added for permeability. The cells were then blocked with 5% goat serum for 1 h and incubated with the primary anti-GRIM-19 antibody (ab110240, 1:1000, Abcam, Cambridge, UK) overnight at 4 ℃. Subsequently, they were incubated with the Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113, 1:200, Abcam, Cambridge, UK) for 1 h at RT. The nucleus was stained with the DAPI solution. The images were observed and collected under a fluorescence microscope.
Ab110240
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab110240 is a primary antibody that targets the protein PD-L1 (Programmed cell death 1 ligand 1). It is intended for use in immunocytochemistry, immunofluorescence, and flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab92547, by Abcam (2 mentions)
Ab14745, by Abcam (2 mentions)
Ab16505, by Abcam (1 mentions)
Ab15580, by Abcam (1 mentions)
Hybond membrane, by Cytiva (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Hyperfilm, by AGFA HealthCare (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Ab8224, by Abcam (1 mentions)
Ab14705, by Abcam (1 mentions)
Ab14734, by Abcam (1 mentions)
Ab117991, by Abcam (1 mentions)
Ab196019, by Abcam (1 mentions)
Ab110249, by Abcam (1 mentions)
Ab290, by Abcam (1 mentions)
Flag hrp, by Merck Group (1 mentions)
Ab19857, by Abcam (1 mentions)
A21422, by Thermo Fisher Scientific (1 mentions)
6 protocols using ab110240
1
Immunofluorescent Localization of GRIM-19
2
Immunohistochemical Profiling of HCC
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After conventional paraffin embedding and sectioning (4 μm), HCC tissue samples were dewaxed with xylene, hydrated with gradient alcohol, and inactivated with 3% H2O2 for 10 min. 0.01 mol/L citrate sodium buffer was applied for microwave repair (pH = 6.0, 15 min). After the sections were blocked with 5% bovine serum albumin (BSA) for 20 min, they were incubated with the antibodies of anti-GRIM-19 (ab110240, Abcam, MA, USA), anti-Ki67 (ab15580, Abcam, MA, USA), anti-E-cadherin (ab16505, Abcam, MA, USA), and anti-Vimentin (ab92547, Abcam, MA, USA) at 4 ℃ overnight. The next day, the goat-anti-rabbit IgG was added and incubated at RT for 20 min. After washing with PBS, DAB was used for color development. After hematoxylin restaining, the sections were dehydrated, transparentized, mounted, and examined under a microscope. Image-Pro Plus (Media Cybem etics, America) was applied for analysis.
3
Western Blot Protein Detection
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Cells were lysed with RIPA Lysis and Extraction Buffer (89901, Thermo Fisher, Waltham, MA, USA) including HaltTM Protease Inhibitor Cocktail (78410, Thermo Fisher) and protein concentrations were determined using the Bradford method (Molecular Devices, Downingtown, PA, USA). Protein samples were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to Hybond membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Proteins were incubated with antibodies against GRIM19 (ab110240, Abcam, Cambridge, MA, USA), phospho-STAT3 (Tyr705; 9145, Cell Signaling, Danvers, MA, USA), phospho-STAT3 (Ser727; 9134, Cell Signaling), STAT3 (Cell 9139, Signaling), and β-actin (sc-47778, Santa Cruz Biotechnology). They were then detected using an enhanced chemiluminescence detection kit (Pierce, Rockford, IL, USA) and HyperFilm (Agfa, Mortsel, Belgium), with β-actin used as a loading control.
4
Mitochondrial Protein Detection Assay
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ATP5F1 (ab117991, Abcam, 1:1000), β-Actin (ab8224, Abcam, 1:1000), COX IV-Alexa Fluor 488 (4853 S, Cell Signaling Tech [CST], 1:200), EMRE (A300-BL19208, Bethyl, 1:1000), FLAG HRP (A8592, Sigma, 1:1000), FLAG magnetic beads (M8823, Sigma), FOXRED1 (sc-377264, Santa Cruz, 1:1000), GAPDH (2118 S, CST, 1:1000), GFP (ab290, Abcam, BN-PAGE: 1:1000), goat anti-mouse Alexa Fluor 555 (A21422, ThermoFisher, 1:100), goat anti-rabbit Alexa Fluor 488 (A32731, ThermoFisher, 1:100), HA (3724 S, CST, 1:800), HA HRP (12013819001, Sigma, 1:1000), MCU (14997 S, CST, WB 1:1000, ICC 1:100, DuoLink 1:200), MICU1 (12524 S, CST, 1:1000), MICU1 (HPA037480, Sigma, 1:1000), MTCO1 (ab14705, Abcam, 1:100), NDUFA13 (ab110240, Abcam, 1:1000), NDUFB10 (ab196019, Abcam, 1:1000), NDUFS2 (ab110249, Abcam, WB 1:1000, ICC 1:100, DuoLink 1:200), NDUFS3 (ab177471, ICC 1:200), NDUFS4 (ab137064, WB 1:1000), NDUFS4 (PA5-21677, ThermoFisher, BN-PAGE: 1:300), Oct4 (ab19857, Abcam, 1:200), ROMO1 (TA505580, Origene, 1:1000), Sox2 (5024, CST, 1:200), TOM20 (42406 S, CST, BN-PAGE: 1:800), VDAC1 (ab14734, Abcam, 1:1000), Vimentin (ab92547, Abcam, 1:1000), ZO-1 (33-9100, ThermoFisher, 1:200).
5
Immunoblotting Analysis of Mitochondrial Proteins
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Total fibroblast cell lysates were subjected to whole protein quantification, separated on 4–12% precast gels (Lonza) by SDS–polyacrylamide gel electrophoresis (PAGE) electrophoresis and semi-dry transferred to polyvinylidene difluoride membranes (GE Healthcare Life Sciences). The membranes were blocked in 5% non-fat milk (Bio Rad) in TBS-T (150 mM NaCl, 30 mM Tris base, pH 7.4, 0.1% Tween 20) for 1 h and immunoblotted using primary antibodies (1:1,000 dilution) against CLPP (Abcam, ab56455), MCOLN1 (Abcam, ab28508), MT-ND5 (Abcam, ab92624), NDUFA13 (Abcam, ab110240), NDUFB3 (Abcam, ab55526), NDUFB8 (Abcam, ab110242), TIMMDC1 (Abcam, ab171978) and UQCRC2 (Abcam, ab14745) for 1 h at RT or ON at 4 °C. Signals were detected by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (Jackson Immuno Research Laboratories, Code: 111-036-045 and Code: 115-036-062, respectively, 1:5,000 dilution) for 1 h and visualized using ECL (GE Healthcare Life Sciences).
6
Blue Native PAGE Analysis of Membrane Proteins
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For blue native PAGE analysis, the solubilization of membrane proteins was performed using 4% digitonin (Sigma-Aldrich) according to the previously described protocol [26] . The separated proteins were transferred onto 0.22-µm polyvinylidene di uoride membranes (Bio-Rad, Hercules, CA, USA) for subsequent experiments. The membranes were then probed with anti-GRIM19 (Abcam, Cambridge, United Kingdom, ab110240, 1:1000), anti-SDHA (Abcam, ab14715, 1:2000), anti-UQCRC2 (Abcam, ab14745, 1:1000), anti-MT-COI (Abcam, ab14744, 1:1000), anti-ATP5A (Abcam, ab14748, 1:2000), anti-TOM70 (Proteintech, IL, USA, 14528-1-AP, 1:2000), and anti-β-actin (Santa Cruz Biotechnology, TX, USA, sc-47778, 1:2000).
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