Confirmed, formalin-fixed, paraffin-embedded human ovarian normal tissue and epithelial tumors were obtained from the Department of Pathology, The Ohio State University (Columbus, OH). Cores were obtained from the most viable/nonnecrosis areas of the tissue. Each sample had 2 independent cores. A tissue microarray (TMA) was constructed from 16 normal tissues and 43 patients with ovarian cancer (grade 1, 4; grade 2, 14; grade 3, 25). TMA sections were subjected to deparaffinization and rehydration. The endogenous peroxidase was quenched by 3% (v/v) hydrogen peroxide. The epitope retrieval was performed using Dako TRS solution (Dako) for 25 minutes at 96°C in a vegetable steamer. Primary mouse antibody against DDB2 (ab51017; 1:10; Abcam) was incubated for 1 hour at room temperature, and detected using a Mach 3 Mouse HRP-Polymer kit (Biocare Medical) and diaminobenzidine teterhydroxychloride (Dako). Tissues were counterstained with Richard Allen hematoxylin. Intensity of staining was blind scored from 1 (no staining) to 4 (highest intensity of staining).
Ab51017
Manufactured by Abcam
Ab51017 is a recombinant antibody product manufactured by Abcam. This product is designed for laboratory use.
Automatically generated - may contain errors
Lab products found in correlation
Ab2356, by Abcam (1 mentions)
Ab97522, by Abcam (1 mentions)
Ab85914, by Abcam (1 mentions)
D1m5y, by Cell Signaling Technology (1 mentions)
Vectashield dapi mounting media, by Vector Laboratories (1 mentions)
Sp5 confocal microscope, by Leica camera (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
3 protocols using ab51017
1
Immunohistochemistry of DDB2 in Ovarian Cancer
2
Immunofluorescence of DNA Repair Proteins
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Immunofluorescence were performed according to standard protocols with the following antibodies: anti-XPC (1:200, D1M5Y, Cell Signaling), anti-XPA (1:200, ab85914, Abcam), anti-CPD (1:50, KTM53, Kamiya Biomedical Company), anti-TFIIH p62 (1:50, Q-19, Santa Cruz Biotech) anti-DDB-1 (1:100, ab97522, Abcam), anti-DDB-2 (10 µg mL−1, ab51017, Abcam), and anti-ERCC1 (1 µg mL-1, ab2356, Abcam). All slides were cover-slipped using Vectashield-DAPI mounting media (Vector laboratories, Burlingame, CA, USA). Images per sample were captured using the Leica SP5 Confocal Microscope at BU Cellular Imaging Core. To determine the relative fluorescence at local irradiated sites, the CPD-positive area was gated by the Image J software and fluorescence intensity was quantified. Then the fluorescence intensity of XPC at the same area was quantified and divided by the CPD fluorescence. Relative fluorescence was calculated as the experimental fluorescence divided by the control fluorescence69 (link).
3
Chromatin Binding Analysis of UV-DDB
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Salt extraction and micrococcal nuclease (MNase) digestion was used to analyze the binding of UV-DDB to chromatin [25] (link). After electrophoretic separation, the samples were transferred to a polyvinylidene (PVDF) membrane (BioRad) blocked by incubation for 2 h at room temperature with Tris-buffered saline containing 0.05% (v/v) Tween-20 and 5% (w/v) nonfat dry milk. Antibodies against the following proteins were used in Western blots: DDB2 (dilution 1∶50, ab51017, Abcam), GAPDH (1∶4′000, No. 4300, Ambion), H3 (1∶10′000, No. 07-690, Millipore). HRP-conjugated secondary antibodies were diluted 10,000-fold. Reactions were developed with SuperSignal West Pico (Pierce), recorded with a FUJI LAS-3000 imaging system and quantified using the Quantity One software (BioRad).
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