Scraped mucosa was homogenized in tissue lysis buffer containing 20 mM Tris (pH 8); 0.15 M NaCl; 2 mM EDTA; 1 mM sodium vanadate; 0.1 M sodium fluoride; 50 mM β-glycerophosphate; 5% glycerol, 2x protease inhibitor (Roche Applied Science); 1% Triton; and 0.1% SDS. Homogenate (60 μg protein) were resolved by 10% SDS-PAGE, transferred to PVDF membrane, and probed with mouse anti-HuR antibody (3A2)(Sc5261 Santa Cruz Biotechnology Inc.), mouse anti-Cyclin D1 (Mouse MAb, NeoMarkers, Fremont, CA), rabbit anti-C-Myc (Sc-764, Santa Cruz Biotechnology Inc.), rabbit anti-OLFM4 (ab96280 Abcam Inc.) and a rabbit anti-β-catenin antibody (9582, Cell Signaling Technology). Equal loading was verified using a rabbit anti-α actin antibody (A2066, Sigma-Aldrich) or rabbit anti-Hsp40 (SPA-400, Assay designs).
Ab96280
Manufactured by Abcam
Sourced in United Kingdom
Ab96280 is a lab equipment product. It is a high-quality product designed for laboratory use.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (1 mentions)
A2066, by Merck Group (1 mentions)
Sc 764, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti β catenin antibody, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase labelled secondary antibody, by Maixin Group (1 mentions)
Mib 1, by Agilent Technologies (1 mentions)
Confirm anti er sp1, by Roche (1 mentions)
Confirm anti pr 1e2, by Roche (1 mentions)
Histofine kit, by Nichirei Biosciences (1 mentions)
Herceptest, by Agilent Technologies (1 mentions)
Ventana benchmark xt, by Roche (1 mentions)
3 protocols using ab96280
1
Protein Extraction and Immunoblotting Protocol
2
Immunohistochemical Analysis of OLFM4, ERα, and PR in FFPE Tissue
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Sections (4 μm) were cut from FFPE tissue blocks, and IHC was conducted as described previously [25 (link)] with antibodies to OLFM4 (ab96280, rabbit anti-human polyclonal, diluted at 1/100; Abcam, Cambridge, England), ERα (Kit-0012-2, rabbit anti-human monoclonal, ready to use; Maixin Biotechnology, Fuzhou, Fujian, China), PR (Kit-0013-2, rabbit anti-human monoclonal, ready to use; Maixin Biotechnology), and horseradish peroxidase-labelled secondary antibody (Maixin Biotechnology) in accordance with manufacturer*s instructions. Colour was developed with diaminobenzidine (Dako Corp, Carpinteria, CA, USA) incubated for 5–10 min. at room temperature. Slides were counterstained with haematoxylin and examined by light microscopy.
The immunoreactive score (IRS) was used to evaluate results: IRS = staining intensity × percent of positive cells. Staining intensity was graded according to the following criteria: 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellow with brown) and 3 (strong staining, brown). The percent staining was graded according to the proportion of positive stained cells as follows: 0 for ≤5% positive cells; 1 for 6–25% positive cells; 2 for 26–50% positive cells and 3 for ≥51% positive cells. An IRS score of 4 and higher is regarded as high expression of genes.
The immunoreactive score (IRS) was used to evaluate results: IRS = staining intensity × percent of positive cells. Staining intensity was graded according to the following criteria: 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellow with brown) and 3 (strong staining, brown). The percent staining was graded according to the proportion of positive stained cells as follows: 0 for ≤5% positive cells; 1 for 6–25% positive cells; 2 for 26–50% positive cells and 3 for ≥51% positive cells. An IRS score of 4 and higher is regarded as high expression of genes.
3
Immunohistochemical Evaluation of Biomarkers
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Rabbit polyclonal antibodies for OLFM4 (ab96280) and LY6D (HPA024755) and mouse monoclonal antibody for S100A7 (47C1068) were purchased from Abcam (Cambridge, UK), Sigma‐Aldrich (St. Louis, MO, USA) and LifeSpan BioSciences (Seattle, WA, USA), respectively. Immunostaining for these 3 antibodies was automatically performed using Ventana Benchmark XT platform (Roche Diagnostics Japan, Tokyo, Japan). As a positive control, we used human tissue of the prostate, skin and skin for OLFM4, LY6D and S100A7, respectively, based on the data sheet (OLFM4) and database of the Human Protein Atlas (https://www.proteinatlas.org ) (LY6D and S100A7).
Immunohistochemistry for ER (CONFIRM anti‐ER [SP1]) and progesterone receptor (PR) (CONFIRM anti‐PR [1E2]; Roche Diagnostics Japan) was also performed with Ventana Benchmark XT (Roche Diagnostics Japan), and that for HER2 was performed by HercepTest (DAKO). Mouse monoclonal antibody for Ki67 (MIB1) was purchased from DAKO (Carpinteria, CA, USA), and a Histofine kit (Nichirei Bioscience, Tokyo, Japan) was used for the immunohistochemistry.
Immunohistochemistry for ER (CONFIRM anti‐ER [SP1]) and progesterone receptor (PR) (CONFIRM anti‐PR [1E2]; Roche Diagnostics Japan) was also performed with Ventana Benchmark XT (Roche Diagnostics Japan), and that for HER2 was performed by HercepTest (DAKO). Mouse monoclonal antibody for Ki67 (MIB1) was purchased from DAKO (Carpinteria, CA, USA), and a Histofine kit (Nichirei Bioscience, Tokyo, Japan) was used for the immunohistochemistry.
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