Cells were lysed in NETN lysis buffer (100 nM Tris-Cl [pH 7.8], 1 mM EDTA, 100 mM NaCl, and 0.1% Triton X-100) with protease and phosphatase inhibitors (Roche). Protein concentration was determined by Lowry protein assay. Protein samples were separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were incubated with antibodies directed against phospho-SMAD1/5/8 (Cell Signaling), total SMAD1/5/8 (Cell Signaling), p63 (4A4 clone; Lab Vision), p53 (Clone DO-7; Thermo Scientific), and β-Actin (Cell Signaling) primary antibodies. Horseradish peroxidase (HRP) conjugated secondary antibodies were used. Blots were visualized by enhanced chemi-luminescence (Millipore).
P53 clone do 7
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The P53 (Clone DO-7) is a monoclonal antibody designed for the detection of the p53 protein. It recognizes an epitope within the N-terminal domain of p53 and can be used for immunohistochemical staining applications.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence, by Merck Group (1 mentions)
Phospho smad1 5 8, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Pierce peroxidase ihc detection kit, by Thermo Fisher Scientific (1 mentions)
2 protocols using p53 clone do 7
1
Western Blot Analysis of Phosphorylated SMAD1/5/8
2
Immunohistochemical Analysis of Oral Lesions
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112 cases were included which comprised of 12 benign cases, 19 premalignant cases (six OIN 1, four OIN II and nine OIN III), 41 cases of well-differentiated squamous cell carcinomas (WDSCC), 31 cases of moderately differentiated SCC (MDSCC), 8 cases of poorly differentiated SCC (PDSCC) and 3 cases of verrucous carcinoma. The selected sections were mounted on poly-L-lysine-coated slides. Immunohistochemistry (IHC) was performed using antibodies Ki-67 (Clone SP6, dilution 1:200, Thermo Fisher Scientific, UK), p-53 (clone DO-7, dilution 1:200, Thermo Fisher Scientific, UK) and p-16 (clone 1E12E10, Thermo Fisher Scientific, UK) as indicated on Pierce™ Peroxidase IHC Detection Kit by Thermo Fisher Scientific. The results were recorded as index of positivity (IP) score and staining intensity in accordance with previous studies.[9 (link)10 (link)] IP score was assigned 0 (<10%), 1 (10%–50%) and 2 (>50%) according to the percentage of positive stained cells in 1000 counted cells in the ×40 microscope field. Only a score of 1 or 2 was considered as positive immunoexpression. 0 score was considered as negative. For p-53 and Ki-67, nuclear positivity was considered as positive expression, and for p-16, combined nuclear and cytoplasmic staining was considered as positive expression. Intensity of staining was scored as 0 (weak), 1 (moderate) and 2 (strong).[9 (link)10 (link)]
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