Mouse oviductal epithelial cells (MOECs) were treated with or without proteins WNT4, RSPONDIN2, or compound CT99021 overnight, and chromatin immunoprecipitation (ChIP) analysis was performed as previously described (Wu et al., 2007 (link)). DNA was precipitated with a polyclonal NR5A2 (Abcam Cat# ab189876) antibody or mouse IgG as a negative control. Precipitated DNA was amplified with the primers designed to the Cyp11a1 and Hsd3b1 promoter regions. See the detailed information of the primers in Supplementary Table 1 . PCR products were confirmed by sequencing.
Ab189876
Manufactured by Abcam
Sourced in United Kingdom
Ab189876 is a primary antibody that recognizes the protein target X. It is suitable for use in various immunoassay applications, including ELISA, Western blotting, and immunohistochemistry.
Automatically generated - may contain errors
5 protocols using ab189876
1
Investigating NR5A2 Binding to Key Genes
2
Immunohistochemical Analysis of LRH-1 in OS
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Human formalin-fixed paraffin-embedded (FFPE) OS tissue microarray slides (OS804C and B024H) were purchased from Xi’an Taibosi Biotechnology Co., Ltd., with core diameters of 1.5 mm and thickness of 5 μm. OS804C contains 40 cases of OS tissue sample (2 cores/case), and clinicopathological characteristics of OS patients were shown in Table 1 . B024H contains 11 cases of normal bone or ribs tissues (2 cores/case). The study was approved by the Ethics Committee of Harbin Medical University Cancer Hospital.
The detailed experimental procedures of tissue dewaxing and antigen retrieval of the slides were described previously (Cheng et al., 2018 (link); Xing et al., 2018 (link)). The slides were stained for LRH-1 immunohistochemistry (IHC) with the corresponding anti-LRH-1 (1:100 dilutions, ab189876; Abcam, Cambridge, United Kingdom). The levels of LRH-1 staining were scored as previously described (Liu et al., 2018 (link)).
The detailed experimental procedures of tissue dewaxing and antigen retrieval of the slides were described previously (Cheng et al., 2018 (link); Xing et al., 2018 (link)). The slides were stained for LRH-1 immunohistochemistry (IHC) with the corresponding anti-LRH-1 (1:100 dilutions, ab189876; Abcam, Cambridge, United Kingdom). The levels of LRH-1 staining were scored as previously described (Liu et al., 2018 (link)).
3
LRH1 Expression Quantification in Tumors
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Resected paraffin‐embedded tissue blocks (~4 μm) were stained with hematoxylin and eosin for tumor confirmation. To enhance immunoreactivity, antigen was retrieved using a pressure cooker with pH 6.0 citrate buffer (10 mM) for three minutes.
Subsequently, LRH1 staining was performed with anti‐LRH1 (1:100 dilutions; ab189876; Abcam, Cambridge, UK) overnight at 4°C. Rabbit secondary antibody was added to the sample and left for 50 minutes at room temperature. The slides were then stained using Mayer's hematoxylin. The positive control contained positive LRH1 expression in human lung carcinoma, while the negative control was stained with rabbit serum. Positive cells were assigned rank by percentage: 0 = 0%, 1 = 1–10%, 2 = 11–50%, 3 = 51–70%, 4 = ≥ 71%. Staining was ranked by intensity: 0 = no staining, 1 = weak staining, 2 moderate staining, and 3 = intense staining. Finally, LRH1 expression was evaluated by the sum of the percentage of positive cells and the intensity of staining. After strict statistical analysis, the level of LRH1 expression was divided into two stages: low < 4 and high ≥ 4. Two pathologists blindly analyzed any discrepancies to avoid bias.
Subsequently, LRH1 staining was performed with anti‐LRH1 (1:100 dilutions; ab189876; Abcam, Cambridge, UK) overnight at 4°C. Rabbit secondary antibody was added to the sample and left for 50 minutes at room temperature. The slides were then stained using Mayer's hematoxylin. The positive control contained positive LRH1 expression in human lung carcinoma, while the negative control was stained with rabbit serum. Positive cells were assigned rank by percentage: 0 = 0%, 1 = 1–10%, 2 = 11–50%, 3 = 51–70%, 4 = ≥ 71%. Staining was ranked by intensity: 0 = no staining, 1 = weak staining, 2 moderate staining, and 3 = intense staining. Finally, LRH1 expression was evaluated by the sum of the percentage of positive cells and the intensity of staining. After strict statistical analysis, the level of LRH1 expression was divided into two stages: low < 4 and high ≥ 4. Two pathologists blindly analyzed any discrepancies to avoid bias.
4
Immunohistochemical Analysis of Oviduct
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Oviducts were collected and fixed in Bouin’s solution for hematoxylin and eosin (H&E) staining and in 4% paraformaldehyde (PFA) for immunohistochemistry (IHC). For IHC assay, sections (4–5 μm) were deparaffinized in xylene and rehydrated in gradient alcohol concentrations. After antigen retrieval, the sections were blocked by 5% bovine serum albumin (BSA). The sections were then incubated with primary antibodies overnight at 4°C. The next day, the sections were incubated with secondary antibodies for 20 min and then developed with 3,3′-diaminobenzidine and counterstained with hematoxylin. Antibodies were diluted as follows: OVGP1 at 1:100 (Santa Cruz, sc-377267), PAX8 at 1:100 (Proteintech Cat# 60145-4-lg), CTNNB1 at 1:100 (BD Transduction LaboratoriesTM, Cat# 610153), NR5A1 at 1:200 (Abcam Cat# ab217317), and NR5A2 at 1:500 (Abcam Cat# ab189876). Samples from at least three animals were evaluated to ensure reproducibility of the results.
5
Quantitative Protein Expression Analysis
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Western blotting was performed by following a routine protocol. Briefly, LCs were washed with phosphate buffered saline (PBS) and scraped into 200 μL sodium dodecyl sulphate (SDS) electrophoresis sample buffer. Cell lysates were then sonicated (60 Hz, 10 s for 3 times) and heated for 10 min at 95°C. Protein samples were separated by 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and were then transferred to a nitrocellulose membrane (NC membrane). The blotting membranes were blocked with 5% w/v non-fat milk and probed with rabbit anti-SF1 (1:800 dilution, Cell Signaling Technology, D1Z2A), rabbit anti-LRH1 (1:800 dilution, Abcam, ab189876), rabbit anti-CREB (1:1000 dilution, Cell Signaling Technology, 48H2), rabbit anti-CREM (1:1000 dilution, Abcam, ab64832) and rabbit anti-CYP19 (1:400 dilution, Santa Cruz Biotechnology, sc-30086) respectively. After being washed, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:1000 dilution, Sigma-Aldrich, A0545) for 1 h at room temperature. The bound antibodies were visualized by a chemiluminescence system (BIO-RAD Biosciences). The optical densities of the bands were quantified by using Image J software (NIH). GAPDH served as loading control.
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