Liver tissues were fixed with 4% paraformaldehyde solution, embedded in paraffin blocks and processed by immunohistochemistry staining. Tissue sections were deparaffinized and rehydrated using a graded ethanol series and distilled water, and then treated with 3% H2O2 in methanol for 30 min to block endogenous peroxidase activity. Tissue sections were then rinsed twice for five minutes in phosphate-buffered saline (PBS) and incubated with 10% normal goat serum for 30 min to block non-specific antibody binding. After washing, the samples were incubated with primary antibodies against CYP7A1 (Abcam, ab234982, 1:500), CYP8B1 (Abcam, ab175843, 1:50), CYP27A1 (Abcam, ab126785, 1:250), and CYP7B1 (Abcam, ab175889, 1:100). Sections were then washed in PBS three times and incubated with secondary antibodies. The sections were stained with DAB according to the manufacturer’s protocol, mounted on slides, and photographed using a digital microscope camera (Nikon, Tokyo, Japan). The immunohistochemistry sample images were quantified using Image-Pro Plus software (Media Cybernetics, MD, USA).
Ab126785
Manufactured by Abcam
Sourced in United States
Ab126785 is a reagent used for cell culture experiments. It is a monoclonal antibody that targets a specific protein in mammalian cells. The core function of this product is to enable researchers to detect and quantify the target protein in their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ab175843, by Abcam (2 mentions)
Ab181616, by Abcam (2 mentions)
Ab15580, by Abcam (2 mentions)
Ripa lysis and extraction buffer, by Solarbio (2 mentions)
Ab8227, by Abcam (2 mentions)
Ab32072, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Digital microscope camera, by Nikon (1 mentions)
Ab234982, by Abcam (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Nupage lds sample buffer and reducing agent, by Thermo Fisher Scientific (1 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Ecl western blotting kit, by Thermo Fisher Scientific (1 mentions)
Bs 12867r, by Bioss Antibodies (1 mentions)
Ab92494, by Abcam (1 mentions)
Ab51495, by Abcam (1 mentions)
Ab32147, by Abcam (1 mentions)
Ab32049, by Abcam (1 mentions)
Ab109520, by Abcam (1 mentions)
9 protocols using ab126785
1
Immunohistochemical Analysis of Liver Cytochrome Enzymes
2
Quantitative Western Blot Analysis
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For electrophoresis, cell lysate samples were denatured with NuPAGE LDS sample buffer and reducing agent (Thermo Fisher Scientific, Waltham, MA, USA) at 70°C for 10 min and run on 10% Bis-Tris precast gels (Thermo Fisher Scientific, Waltham, MA, USA) using NuPAGE MOPS SDS running buffer at 150 V for 1h20 min. For immunostaining and after gel electrophoresis, the proteins were transferred onto a PVDF membrane via semi-dry Western blotting at 25V for 2 h, blocked for 1 h at room temperature with 5% BSA in TBS-T (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Tween 20) and stained with a monoclonal antibody against CYP27A1 (ab126785, Abcam, Cambridge, UK) diluted 1:1000, followed by the secondary anti-rabbit HRP-conjugated antibody (W401B, Promega), diluted 1:10.000 in 5% BSA TBS-T. After each incubation, the membrane was washed 3 times for 5 min with TBS-T, and finally developed with Clarity Western ECL substrate (Bio-Rad) for 5 min.
3
Western Blot Analysis of MTDH, PTEN, and AKT
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Total protein was isolated from tissue samples, cells, or tumor xenografts using radioimmunoprecipitation assay (RIPA) Lysis and Extraction Buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Proteins were denatured, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in a 10% gel, and transferred onto polyvinylidene fluoride membranes. After blockage with 5% fat-free dried milk diluted in Tris-buffered saline (TBS) containing 0.1% of Tween 20 for 2 h, the membranes were incubated overnight at 4 °C with the following primary antibodies: anti-MTDH (cat. # ab126785; dilution 1:500; Abcam, Cambridge, MA, USA), anti-PTEN (ab32072; 1:500; Abcam), anti-phospho- (p-)AKT (ab181616; 1:500; Abcam), anti-AKT (ab15580; 1:500; Abcam), and anti-GAPDH (ab8227; 1:500; Abcam). A horseradish peroxidase–conjugated secondary antibody (dilution 1:3,000; cat. # 7075S, Cell Signaling Technology, Inc., Danvers, MA, USA) was incubated with the membranes at room temperature for 2 h. The Enhanced Chemiluminescence (ECL) Western Blotting Kit (Pierce Biotechnology Inc., Rockford, IL, USA) was employed for visualization of the protein signals.
4
Immunohistochemical Analysis of Liver and Ileum
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The frozen sections of the liver and ileum was fixed with 10% paraformaldehyde for 15 min. The sections were permeableised in 0.25% Triton X-100 and then blocked with 5% goat serum for 30 min. After incubation with primary antibody overnight at 4 °C and secondary antibody for 1 h at RT, the slides were washed thrice with PBS and then counterstained using DAPI. The following primary antibodies were used in this study: anti-NR1H4 antibody (Bioss, bs-12867R; 1:200); anti-CYP7A1 antibody (Bioss, bs-21430R, 1:200); and anti-CYP27A1 antibody (Abcam, ab126785, 1:200). The images were obtained by fluorescence microscopy.
5
Western Blot Analysis of Protein Expression
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Following transfection, cells and tissues were lysed for 48 h using RIPA Lysis and Extraction Buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and the protein concentration was measured using Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Following heating at 100°C for 10 min in the presence of a loading buffer, equal amounts of protein lysates (50 µg) were separated using 10% SDS-PAGE (Bio-West Inc., Logan, UT, USA) at 100 V for 1 h, and transferred onto Invitrogen nitrocellulose membranes (Thermo Fisher Scientific, Inc.) at 120 V for 1 h. Following blocking with 5% skimmed milk (diluted with PBS), the membranes were incubated overnight at 4°C with the following primary antibodies: CYP27A1 (ab126785; 1:500; Abcam, Cambridge, MA, USA), c-myc (ab32072; 1:500; Abcam), RB (ab181616; 1:500; Abcam), Ki-67 (ab15580; 1:500; Abcam), CDK2 (ab32147; 1:500; Abcam), p21 (ab109520; 1:500; Abcam), p53 (ab32049; 1:500; Abcam), PDCD4 (ab51495; 1:500; Abcam), SOX2 (ab92494; 1:500; Abcam), β-actin (ab8227; 1:500; Abcam). Subsequently, the membranes were incubated with secondary goat monoclonal (RMG01) to rabbit IgG Fab region (Biotinylated; ab222772; 1:10,000; Abcam) at room temperature for 2 h, and proteins were detected using enhanced chemiluminescence (Pierce™ ECL Western Blotting Substrate; Thermo Fisher Scientific, Inc.).
6
Immunohistochemical Analysis of CYP27A1
7
Hepatic Protein Analysis via WES
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Total protein was extracted as described in detail (Xie et al., 2016 (link)), and WES was performed as previously described (Zhou et al., 2019 (link)). Briefly, hepatic protein lysates were mixed with 5 × fluorescent master mix and boiled for 5 min. Then, the samples, wash buffer, primary antibodies including phosphorylated protein kinase B (P-AKT; #9271; Cell Signaling Technology), protein kinase B (AKT; #9272; Cell Signaling Technology), phosphorylated hormone-sensitive lipase (P-HSL; ab109400; Abcam), hormone-sensitive lipase (HSL; ab45422; Abcam), sterol regulatory element-binding transcription factor 1c (SREBP-1c; ab28481; Abcam), cytochrome P450 family 27 subfamily a member 1 (CYP27A1; ab126785; Abcam), GAPDH (# 2118; Cell Signaling Technology), secondary antibodies, blocking reagent, and then, in a manufacturer-provided microplate, chemiluminescent substrate was dispensed into the designated wells. In the individual capillaries, protein separation was performed automatically using the default settings. Compass software 3.1 (Protein Simple, San Jose, CA, USA) was used to analyze the data.
8
Western Blot Protocol for Protein Detection
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Whole cell lysates were derived from cells or tissues using radioimmunoprecipitation assay (RIPA) lysis buffer. Proteins (20–40 μg) with SDS-loading buffer were loaded onto gels and run on SDS-PAGE; then, proteins were transferred to the polyvinylidene fluoride (PVDF) membrane. The PVDF membranes were incubated with primary antibodies against Pro-caspase-1 (1:2000, #ab179515, Abcam), caspase-1 (1:2000, #ab108362, Abcam), NLRP3 (1:2000, #ab16097, Abcam), IL-1β (1:1000, #AF-401-NA, R&D), HO-1 (1:500, #sc-10789, Santa Cruz), GAPDH (1:500, #sc-47724, Santa Cruz), β-actin (1:500, #sc-81178, Santa Cruz), Tubulin (1:1000, #556321, BD Pharmingen), FXRa (1:2000, #ab187735, Abcam), Cyp8b1 (1:2000, #ab175843, Abcam), Cyp27a1 (1:1000,#ab126785, Abcam), Sult2a1 (1:500, #sc-376629, Santa Cruz), Bsep (1:2000, #ab155421, Abcam), Mrp3 (1:500, #sc-5776, Santa Cruz), Ntcp (1:2000, #ab131084, Abcam), and BAAT (1:2000, #ab83882, Abcam). Then, appropriate horseradish peroxidase (HRP)-linked secondary antibodies were incubated for further 1 hour at room temperature. Luminal and H2O2 substrates were used for chemiluminescence detection.
9
Tumor-Specific CYP27A1 Expression Analysis
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The tumor cell-specific expression of CYP27A1 was determined by immunohistochemistry following a previously validated protocol (Nelson et al. 2013) . Sections of 3 to 4 µm were cut from whole tissue FFPE blocks, de-paraffinized, treated with antigen retrieval buffer (citrate, pH 6) for 20 min, and then reacted with an anti-CYP27A1 rabbit monoclonal antibody (ab126785, Abcam) at a dilution of 1:500 for 2 h. Staining procedures were performed using the DAKO Envision horseradish peroxidase rabbit/mouse kit (DAKO) and the Dakocytomation Autostainer (DAKO). Cell nuclei were counterstained with hematoxylin. CYP27A1 positivity was detected as a granular cytoplasmic reactivity. Cell type identification and staining intensity was assessed by a board certified pathologist (DG). Only tumor cell-specific CYP27A1 expression was considered for subsequent analyses. Each sample was given a semiquantitative intensity score: 0 (absent), 0.5 (borderline), 1 (weak), 2 (moderate) or 3 (strong). For statistical analysis, the tumors were categorized as negative (0), weak (0.5, 1) and overexpressed (2, 3). Representative cases of CYP27A1 expression in these categories are illustrated in Supplementary Fig. 4.
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