A non-biotin-amplified method (NovoLinkTM Polymer Detection System, Novocastra Laboratories, Newcastle Upon Tyne, UK) was used for visualizing antigens in tissue sections. The sections were subsequently incubated with primary anti-E-cadherin (clone 32A8, Cell Signaling Technology, Beverly, MA, USA), anti-IL-6 (Sigma-Aldrich) and anti-p53 (Novocastra) antibodies diluted in 1% bovine serum albumin in Tris buffer saline overnight at 4 °C using the appropriate dilutions. The slices were then counterstained with hematoxylin, dehydrated and coverslipped. Nuclear p53 immunostaining was assessed by image cytometry, using the Cytometrica program (C&V, Bologna, Italy). Staining was expressed as the percentage of labeled nuclear area over the total nuclear area of epithelial cells in the section labeling index according to Faccioli et al.34
Anti p53
Manufactured by Leica
Sourced in United States, United Kingdom
Anti-p53 is a laboratory reagent used to detect the presence and measure the levels of the p53 protein in biological samples. The p53 protein is a tumor suppressor that plays a crucial role in cell cycle regulation and apoptosis. Anti-p53 is a specific antibody that binds to the p53 protein, allowing its identification and quantification through various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (3 mentions)
Anti actin hrp, by Abcam (2 mentions)
Anti non phosphorylated β catenin, by Cell Signaling Technology (2 mentions)
Anti p erk, by Cell Signaling Technology (2 mentions)
Anti p21, by Santa Cruz Biotechnology (2 mentions)
Anti fibronectin, by Abcam (2 mentions)
Anti n cadherin, by Merck Group (2 mentions)
Anti β catenin, by Cell Signaling Technology (2 mentions)
Anti cox 2, by Thermo Fisher Scientific (2 mentions)
Novolink polymer detection system, by Leica (1 mentions)
Anti il 6, by Merck Group (1 mentions)
Anti e cadherin clone 32a8, by Cell Signaling Technology (1 mentions)
0.45 μm nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti p53 pser15, by Cell Signaling Technology (1 mentions)
Anti p egfr, by Cell Signaling Technology (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti akt and anti pakt, by Cell Signaling Technology (1 mentions)
Anti egfr, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
14 protocols using anti p53
1
Immunohistochemical Analysis of E-cadherin, IL-6, and p53
2
Western Blot Analysis of Cellular Proteins
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Cells or tumors were lysed with RIPA buffer (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS). Samples were separated on a 10% acrylamide gel and transferred to a 0.45 μm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked in TBS containing 5% non-fat milk and 0.05% Tween 20 solution and incubated with primary antibody (Ab). The following antibodies were used for western blot: anti-Zta (Argene, Verniolle, France), anti-p53 (Novocastra, Buffalo Grove, IL, USA), anti-p53 pSer15 (Cell Signaling Technology, Beverly, MA, USA), anti-ATM (Cell Signaling Technology), anti-ATM pSer1981 (Cell Signaling Technology), and anti-β-actin (Sigma-Aldrich)
3
Adriamycin-Induced p53 Expression in Small Intestine Organoids
4
Western Blot Analysis of Protein Expression
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Proteins from whole cell extracts were detected by Western blot according to standard protocols. Primary antibodies: anti-EGFR, anti-pEGFR, anti-ERK1/2, anti-pERK, anti-AKT and anti-pAKT (Cell Signaling Technology), anti-p53 (Novocastra), anti-p21 (Pharmingen), anti-actin (Sigma-Aldrich). Secondary antibodies: anti-mouse and anti-rabbit antibodies (LI-COR Biosciences). The Odyssey® CLx Infrared Imaging System (LI-COR Biosciences) was used for signal detection. Relative signal intensities were given as the quotient of [phospho-protein / unphosphorylated proteins]. Cetuximab-treated samples were normalized to untreated ones and erlotinib-treated samples to DMSO-treated ones.
5
Immunohistochemical Analysis of Signaling Pathways
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Immunohistochemistry (IHC) was performed on a Ventana Benchmark XT autostainer using the XT UltraView diaminobenzidine kit (Ventana Medical Systems, Oro Valley, AZ, USA). The primary antibodies were as follows: monoclonal anti-pERK1/2 antibody (rabbit anti-phospho-p44/42 MAPK [Thr202/Tyr204] clone 20G11, Cell Signaling Technology, Danvers, MA, USA), anti-pAKT (Santa Cruz, sc-135650, 1:30, heat mediated antigen retrieval) and anti-p53 (Novocastra, DO-7, 1:200, heat mediated antigen retrieval).
6
Adriamycin-Induced p53 Expression in Small Intestine Organoids
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Small intestine organoids were grown in 300ul of Matrigel in 1 well each of a 6-well dish containing 3 mls of growth media for 4 days post-passage, then treated with 125ng/ml Adriamycin for 4 hrs to induce p53 expression. Organoids were then recovered from the Matrigel using several rinses with cold PBS. Organoid pellets were lysed with Lamelli buffer. Antibodies used for Western blot were: anti-APC (1:400, FE9 clone, Millipore #MABC202), anti-non-phosphorylated β-catenin (1:1000, #8814, Cell Signaling Technology), anti-p53 (1:500, #NCL-p53-505, Novocastra) and anti-actin-HRP (1:10,000, #ab49900, Abcam).
7
Immunohistochemical Analysis of Liver Markers
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The livers were fixed in phosphate‐buffered 4% paraformaldehyde for 24 hours at 4℃, and paraffin sections were prepared. Immunohistochemical analysis was performed using an EnVision/HRP system (DAKO) on deparaffinized sections treated with target retrieval solution (DAKO). The following antibodies were used: anti‐p53 (Novocastra, Leica Microsystems), anti–cytokeratin 19 (CK19) (provided by Dr Atsushi Miyajima, Institute for Quantitative Biosciences, The University of Tokyo), anti‐Hes1 (Cell Signaling Technology), anti‐YAP (kindly provided by Dr Hiroshi Nishina, Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University), anti–α‐fetoprotein (AFP) (Proteintech), anti–insulin‐like growth factor 2 (IGF2) (Abcam), anti–delta‐like 1 protein (DLK1) (Medical & Biological Laboratories), anti–5‐hydroxymethylcytosine (5hmC; Active Motif), anti‐Myc (Abcam), anti–phosphorylated ERK (Cell Signaling Technology), and ant–Ki‐67 (Nichirei). The chromogen 3,3’‐diaminobenzidine tetrahydrochloride was used to detect the signal (Vector Laboratories), and the sections were counterstained with hematoxylin.
8
Western Blotting Antibody Validation
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The following antibodies were used for Western blotting experiments: anti–Bcl-xL (CST, #2764; 1:1,000), anti-p53 (Novocastra, NCL-p53-505; 1:1,000), anti-Hsp90 (BD, 610418; 1:10,000 to 1:15,000), anticleaved caspase 3 (CST, #9661; 1:1,000), anti-Bad (CST, #9292; 1:1,000), and anti-p21 (Santa Cruz, sc-6246).
9
Protein Extraction and Immunoblotting
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Protein extraction and immunoblotting analysis were performed as previously described by Velaithan et al. 23 Primary antibodies used were anti-MSH2, anti-MLH1, and antihuman PARP (BD Pharmingen, Franklin Lakes, NJ); antiγ-H2A.X (S139), anti-cdc2, anti-p-cdc2 (Y15), anti-p-cdc2 (T14), anti-p-cdc2 (T161), anti-cleaved caspase 3, and anti-p21 Waf1/Cip1 (Cell Signaling, MA); anti-β-actin (Merck Millipore, Palo Alto, CA); and anti-p53 (Novocastra, NE, UK). anti-β-actin monoclonal antibody was used to normalize loading.
10
Comprehensive Protein Expression Analysis
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Standard Western blot assays were used to analyze protein expression. The following antibodies were used for assays: anti-Flag-M2 (Sigma-Aldrich, F1804), anti–β-Actin (Sigma-Aldrich, A5441), anti-HA (Roche, 3F10), anti-His (Santa Cruz Biotechnology, sc-803), anti-GST (Santa Cruz Biotechnology, sc-138), anti-human p53 (Santa Cruz Biotechnology, sc-126), anti-TRIM21 (Abcam, ab207728), anti-MDM2 (2A-10) (66 (link)), anti-Ub (Santa Cruz Biotechnology, sc-8017), and anti-p53 (Leica Biosystems, CM5). All intensity quantification for Western blot was performed by using ImageJ software (NIH).
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