Ab201460

Manufactured by Abcam

Sourced in China

Ab201460 is a laboratory equipment product manufactured by Abcam. It is designed for use in scientific research applications.

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Lab products found in correlation

8 protocols using ab201460

HNF4-Alpha Immunohistochemistry for NASH

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This protocol was approved by the University Health Network Institutional Review Board. Twelve NASH patients were included for HNF4A IHC validation using anti HNF4-Alpha, mouse monoclonal antibody (Abcam, ab201460, rabbit monoclonal, clone EPR16885-99). FFPE sections were (5um) pre-treated for antigen retrieval following manufacturer’s instruction. The dilution for HNF4-alpha antibody was 1:2000 and an anti-rabbit was used as the secondary antibody. The complex was then visualized with hydrogen peroxide substrate and 3, 3’- diaminobenzidine tetrahydrochloride (DAB) chromogen. The slides were then counterstained with Harris Hematoxylin. The degree of HNF4-Alpha staining in each NASH case and healthy liver (control) was assessed using Imagescope software. Entire slides were digitally scanned by an Aperio ScanScope CS scanning system and analyzed by Aperio ImageScope Viewer software (Aperio Technologies Inc., Vista, CA) using the Positive Pixel Count v9 algorithm.

Baciu C., Pasini E., Angeli M., Schwenger K., Afrin J., Humar A., Fischer S., Patel K., Allard J, & Bhat M. (2017). Systematic integrative analysis of gene expression identifies HNF4A as the central gene in pathogenesis of non-alcoholic steatohepatitis. PLoS ONE, 12(12), e0189223.

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Immunohistochemical Analysis of Liver IR

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The specimens were fixed in 4% neutral buffered formalin and then embedded in paraffin. 4 μm-thick Liver sections were stained with hematoxylin and eosin. The severity of liver IR was graded using Suzuki’s criteria on a scale from 0 to 4, or with antibodies using standard immunohistochemistry protocols. For Immunofluorescence, the fixed tissue sections were placed in a repair kit (Servicebio, China) filled with EDTA antigen retrieval buffer (PH = 9.0), and then boiled in a microwave for repair. After natural cooling, the slides were washed three times with PBS. BSA (3%) in PBS solution was added for 30 min to block nonspecific binding, and then the corresponding primary antibodies were added: anti-SGK1(23394–1-AP, Proteintech), anti-p-STAT3 (Tyr705) (bs-1658R, Bioss), anti-HNF-4α (ab201460, Abcam), anti-IL-6 (Servicebio, China), anti-MPO (ab208670, Abcam), anti-CitH3 (ab281584, Abcam), anti-CD36 (Servicebio, China), anti-Ly6G (Servicebio, China), and anti-SAA (Servicebio, China). After overnight incubation, the secondary antibody against the corresponding species was added for 50 min in the dark. DAPI was used for the nuclear counterstaining. Slides were observed under a confocal fluorescence microscope (NIKON ECLIPSE C1) [35 (link)].

Li X., Wang Z., Jiao C., Zhang Y., Xia N., Yu W., Chen X., Wikana L.P., Liu Y., Sun L., Chen M., Xiao Y., Shi Y., Han S, & Pu L. (2023). Hepatocyte SGK1 activated by hepatic ischemia-reperfusion promotes the recurrence of liver metastasis via IL-6/STAT3. Journal of Translational Medicine, 21, 121.

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Immunohistochemical Analysis of Liver Biomarkers

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Immunohistochemistry was performed on FFPE specimens. For antigen retrieval, paraffin sections (3 µm thick) were autoclaved using a microwave for 15 min by adding Tris/EDTA pH 9.0. Anti‐HNF1a (1:3000 dilution; ab242140, Abcam), anti‐HNF4a (1:3000 dilution; ab201460, Abcam), and anti‐CLDN8 (1:2000 dilution, ab183738, Abcam) antibodies were used as the primary antibodies. The staining findings were evaluated by 2 pathologists (ST and YN).

Ishihara H., Yamashita S., Liu Y., Hattori N., El‐Omar O., Ikeda T., Fukuda H., Yoshida K., Takagi T., Taneda S., Kondo T., Nagashima Y., Tanabe K, & Ushijima T. (2020). Genetic and epigenetic profiling indicates the proximal tubule origin of renal cancers in end‐stage renal disease. Cancer Science, 111(11), 4276-4287.

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Examining Targeted Cancer Therapy Mechanisms

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Ribociclib (S7440) and Infigratinib (S2183) were purchased from Selleck Chemicals and were dissolved in 21% Captisol and 30% PEG300 solution (vehicle) for oral administration. Antibodies against FGFR1 (#9740), FGFR3 (#4574), FGFR4 (#8562), AKT (#9272), Rb (#9313), cyclin B1 (#4138), Cdc25C (#4688), Cyclin D1 (#2978), Cyclin E2 (#4132), survivin (#2803), Sox9 (#82630), cleaved PARP (#5625), β catenin (#8480), α‐tubulin (#2144) and phosphorylation‐specific antibodies against AKT Ser473 (#9271), FRS2‐α Tyr439 (#3861), Rb Ser807/811 (#9308), Histone 3 Ser10 (#9701), Cdc2 Tyr15 (#9111) and Erk1/2 Thr202/Tyr204 (#4370) were obtained from Cell Signaling Technology. Antibodies against CYP3A4 (ab124921) and HNF4‐α (ab201460) were from Abcam. The antibodies against FGFR2 (#sc‐122), Erk1/2 (#sc‐94), FRS2‐α (#sc‐17841), GAPDH (sc‐166545) and Cdk2 Thr14/Tyr15 (#sc‐28435‐R) were from Santa Cruz Biotechnology Inc. Anti‐mouse CD31 antibody (#2502) was from BioLegend. Anti‐albumin (#SAB4200711; clone HAS‐11) antibody was purchased from Sigma‐Aldrich.

Prawira A., Le T.B., Vu T.C, & Huynh H. (2020). Ribociclib enhances infigratinib‐induced cancer cell differentiation and delays resistance in FGFR‐driven hepatocellular carcinoma. Liver International, 41(3), 608-620.

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Immunohistochemical Analysis of Tumor Markers

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Immunohistochemistry (IHC) was performed as previously described [20 (link)]. In brief, the sections of tumors were incubated with the following antibodies: HNF4α (ab201460, Abcam), CK-19 (ab52625, Abcam), PCNA (2586, Cell Signaling Technology), p-AKT (Ser473, 4060, Cell Signaling Technology), p-ERK (Thr202/Tyr204, 4370, Cell Signaling Technology), p-NF-κB (Ser536, 3033, Cell Signaling Technology), β-catenin (8480, Cell Signaling Technology), TGF-β (21898-1-AP, Proteintech) and FGFR2 (13042-1-AP, Proteintech).

Xu R.C., Wang F., Sun J.L., Abuduwaili W., Zhang G.C., Liu Z.Y., Liu T.T., Dong L., Shen X.Z, & Zhu J.M. (2022). A novel murine model of combined hepatocellular carcinoma and intrahepatic cholangiocarcinoma. Journal of Translational Medicine, 20, 579.

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Immunofluorescence Assay for Hepatic Progenitors

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On the day of base editing, hepatic progenitors were replated in chamber slides (Thermo Fisher Scientific, 177380PK). Upon completion of the differentiation, cells were fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were permeabilized with 0.3% Triton, then blocked with 5% normal donkey serum, before incubation with primary antibodies overnight at 4°C. Antibodies used were HNF4A (Abcam, ab201460), 2C1 (a kind gift from David Lomas and Elena Miranda), and BiP (Invitrogen, PA1-014A). Following incubation, cells were washed and incubated with appropriate secondary antibodies (Invitrogen, A21202, A21247, and A10042). Finally, nuclei were stained with Hoechst 33342 (Invitrogen, H3570). Cells were imaged using a Leica SP5 confocal microscope, and images were processed in ImageJ and Fiji.

Werder R.B., Kaserman J.E., Packer M.S., Lindstrom-Vautrin J., Villacorta-Martin C., Young L.E., Aratyn-Schaus Y., Gregoire F, & Wilson A.A. (2021). Adenine base editing reduces misfolded protein accumulation and toxicity in alpha-1 antitrypsin deficient patient iPSC-hepatocytes. Molecular Therapy, 29(11), 3219-3229.

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Multiparametric Analysis of Liver Injury

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Reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. Cell culture reagents and media were obtained from Invitrogen Corporation (Carlsbad, CA). The antibodies against α-SMA (ab5694), collagen type I alpha 1 (Col1a1, ab21286), desmin (ab185033), E-cadherin (ab11512), HNF4α (ab201460), VEGFA (ab52917), vimentin (ab92547) and cyclin-dependent kinase inhibitor 1A (p16, ab189043) were purchased from Abcam (Burlingame, CA); cytokeratin-19 (CK-19) antibody was obtained from Developmental Studies Hybridoma Bank (Iowa City, IA); F4/80 and vimentin antibodies were purchased from Cell Signaling (Denver, MA). ELISA kits to measure transforming growth factor-β1 (TGF-β1) levels in serum and cholangiocyte supernatant were purchased from Abcam (Burlingame, CA). RNA was extracted using the mirVana miRNA Isolation Kit from ThermoFisher Scientific and reverse transcribed with the iScript™ cDNA Synthesis Kit from Bio-Rad (Hercules, CA). All primer information for qPCR is listed in Supplemental Table 1.

Zhou T., Kyritsi K., Wu N., Francis H., Yang Z., Chen L., O'Brien A., Kennedy L., Ceci L., Meadows V., Kusumanchi P., Wu C., Baiocchi L., Skill N.J., Saxena R., Sybenga A., Xie L., Liangpunsakul S., Meng F., Alpini G, & Glaser S. (2019). Knockdown of vimentin reduces mesenchymal phenotype of cholangiocytes in the Mdr2−/− mouse model of primary sclerosing cholangitis (PSC). EBioMedicine, 48, 130-142.

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Immunofluorescence Staining of Tissue Sections

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Tissue slides were incubated in PBS containing 0.3% Triton X-100 and 5% BSA for 1 h. The slides were incubated with primary antibodies at 4 °C overnight. After a thorough wash, cells were incubated with the appropriate fluorescence-conjugated secondary antibodies (1:500) for 1 h at room temperature. Finally, the cell nuclei were stained with Hoechst 33342 solution for 30 min. Cells were fixed in 4% PFA at room temperature for 30 min, then follow the same staining protocol as tissue slides. Images were captured with an Olympus IX71 inverted fluorescent microscope and analyzed by professional image analysis software (Image J).
Antibodies used include α-SMA (1:200; Invitrogen MAI-06110), collagen1a1 (1:200; Arigo arg21965), Vimentin (1:200; Abcam ab8978), HNF4α (1:200; Abcam ab201460), CK19 (1:200; Abcam ab52625), F4/80 (1:200; Abcam ab6640), MLKL (1:200; Abgent AP14272B), p-MLKL (1:200; Abcam ab196436), iNOS (1:200; Abcam ab178945) and Cd206 (1:200; Abcam ab64693).

Guo R., Jia X., Ding Z., Wang G., Jiang M., Li B., Chen S., Xia B., Zhang Q., Liu J., Zheng R., Gao Z, & Xie X. (2022). Loss of MLKL ameliorates liver fibrosis by inhibiting hepatocyte necroptosis and hepatic stellate cell activation. Theranostics, 12(11), 5220-5236.

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