IHC and interpretation was carried out as previously described [15 (link)]. IHC was performed on 4 μm TMA sections using a BOND-MAX automated immunostainer and a Bond Polymer Refine Detection kit (Leica Microsystems, Wetzlar, Germany) according to the manufacturer’s instructions. The primary antibodies used were anti-EPHB3 (Novus Biologicals, Littleton, CO, USA; 1B3) and anti-β-catenin (Novocastra Laboratories, Newcastle, UK; 17C2). EPHB3 expression was assessed at the tumor cell membrane. Histo-scores (H-scores; range: 0–300) were obtained by multiplying the intensity score (0 = negative; 1 = weak; 2 = moderate; 3 = strong) and the percentage of positive tumor cells (range: 0–100%). For statistical analyses, TMA sections with H-scores < 40 were defined as negative, and those with H-scores > 40 were defined as positive. β-catenin staining was considered as positive when more than 10% of the tumor cell showed strong nuclear positivity.
Anti β catenin
Manufactured by Leica Biosystems
Sourced in Italy, United Kingdom
Anti-β-catenin is an antibody product used in various laboratory applications. The antibody specifically binds to the β-catenin protein, which plays a crucial role in cellular signaling and adhesion. This product can be utilized in techniques such as Western blotting, immunohistochemistry, and immunocytochemistry to detect and analyze the presence and distribution of β-catenin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bond max, by Leica Microsystems (2 mentions)
Bond polymer refine detection kit, by Leica Microsystems (2 mentions)
Anti coli, by Merck Group (1 mentions)
4 6 diamidine 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Anti e cadherin, by BD (1 mentions)
Mowiol, by Merck Group (1 mentions)
Anti vimentin, by Leica Biosystems (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Anti n cadherin, by Santa Cruz Biotechnology (1 mentions)
Anti α sma, by Merck Group (1 mentions)
Anti ephb2, by R&D Systems (1 mentions)
Ventana benchmark xt, by Leica Microsystems (1 mentions)
Anti cd44, by Leica Biosystems (1 mentions)
L fabp, by Abcam (1 mentions)
Dako omnis, by Agilent Technologies (1 mentions)
Anti smoc2, by OriGene (1 mentions)
5 protocols using anti β catenin
1
Immunohistochemical Analysis of EPHB3 and β-catenin in Tumor Samples
2
EMT Marker Expression in PDAC Cells
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The expression of key EMT markers and their localization were assessed by immunofluorescence in PDAC cells grown on 12 mm-diameter rounded coverslips coated with FN, LAM, COL-I (Neuvitro Corporation, Vancouver, WA, USA), or uncoated. When they were at the desired confluence, cells were washed in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS-containing 2% sucrose for 10 min at room temperature, post-fixed in 70% ethanol, and stored at −20 °C until use. Cells were incubated with the primary antibodies anti-E-cadherin (1:2500, Becton Dickinson, Milan, Italy), anti β-catenin (1:500, Novocastra, Newcastle upon Tyne, UK), anti-N-cadherin (1:200, Santa Cruz Biotechnology, Heidelberg, Germany), anti-COL-I (1:2000, Sigma-Aldrich, Milan, Italy), anti-vimentin (1:200, Novocastra), and anti-αSMA (1:400, Sigma-Aldrich). Secondary antibodies conjugated with Alexa 488 (1:500, Molecular Probes, Invitrogen, Waltham, MA, USA) were applied for 1 h at room temperature in PBS. Negative controls were incubated omitting the primary antibody. Finally, after incubation with 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) (1:100,000, Sigma-Aldrich), the coverslips were mounted on glass slides using Mowiol.
3
Immunohistochemical Analysis of EPHB2, CD44, and β-catenin
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Immunohistochemistry was performed on 4-μm TMA sections using a Ventana BenchMark XT Staining systems (Leica Microsystems, Wetzlar, Germany) according to the manufacturer’s instructions. The primary antibodies used were anti-EPHB2 (1:700, R&D Systems, Minneapolis, MN, USA), anti-CD44 (1:100, Novocastra Laboratories Ltd., Newcastle upon Tyne, UK), and anti-β-catenin (1:800, 17C2, Novocastra Laboratories). The expression of EPHB2 and CD44 was determined by examining the tumor cell membrane. For each tumor, the intensity and percentage of tumor cells expressing EPHB2 (n = 567) or CD44 (n = 87) were evaluated. Histoscores (H-scores) were calculated by multiplying the intensity score (0, negative; 1, weak; 2, moderate; and 3, strong) and percentage of positive tumor cells (range, 0 to 100), ranging from 0 to 300. For statistical analyses for EPHB2, we used a cutoff of 40 on the basis of the distribution of the H-scores (median value, 40). CRCs with H-score of 40 or lower were classified as negative, while cases with H-score higher than 40 were classified as positive. β-Catenin staining was considered positive when more than 10% of tumor cell nuclei were strongly stained.
4
Histological Evaluation of Liver Samples
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Liver biopsy or surgical sample were formalin-fixed and paraffin-embedded. Hematoxylin-eosin (HE) and Masson’s trichrome stains to assess fibrosis were routinely made. Immunohistochemistry (IHC) with anti-β-catenin (Novocastra, Buccinasco (MI), Italy NCL-L-B-CAT, 1:150 for 30 min), anti-glutamine synthetase (GS-6, Millipore, Limerick, PA, USA, MAB302, 1:400 for 25 min), anti-glypican3 (Gly3, BioMosaics, Santa Barbara, CA 93111 USA B0025R, B0055R, 1:1000 for 30 min), anti-liver fatty acid bending protein (LFABP, Abcam, Cambridge, UK ab 7807, 1:100 for 30 min), and anti-SOX9 (Cell Signaling Technology, Danvers, MA, USA, #82630, 1:100 for 30 min) were performed using Dako Omnis, Agilent Technology (Santa Clara, CA, USA).
5
Immunohistochemical Analysis of SMOC2 and β-Catenin in CRC
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Immunohistochemistry for SMOC2 and β-catenin and were performed on 4-μm TMA sections using a BOND-MAX automated immunostainer and a Bond Polymer Refine Detection kit (Leica Microsystems, Wetzlar, Germany) according to the manufacturer’s guidelines. The primary antibodies used were anti-SMOC2 (OriGene, Rockville, MD; 1:30, catalog number: TA351730) and anti-β-catenin (Novocastra Laboratories, Newcastle, UK; 17C2; 1:800). The expression of SMOC2 was determined by evaluating the whole field of each tumor core (4 mm in diameter). The intensity and percentage of tumor cells expressing SMOC2 in the cytoplasm were examined. Histoscores (H-scores) were measured by multiplying the intensity score (0 = negative; 1 = weak; 2 = moderate; 3 = strong) and percentage of positive tumor cells (range = 0–100), ranging from 0 to 30021 (link). For statistical analyses, we set a cutoff of 40 on the basis of the distribution of H-scores (mean value: 32.5). CRCs with H-score < 40 were classified as negative, while cases with H-score > 40 were classified as positive. Nuclear β-catenin staining was defined as positive when more than 10% of the cancer nuclei were stained for β-catenin. Immunohistochemical analysis was performed blinded to all other data.
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