Bromodeoxyuridine/BrdU Antibody

Bromodeoxyuridine/BrdU in MCF‑7 Human Cell Line. Bromodeoxyuridine/BrdU was detected in immersion fixed MCF-7 human breast cancer cell line stimulated with BrdU using Mouse Anti-Bromodeoxyuridine/ BrdU Antigen Affinity-purified Monoclonal Antibody (Catalog # MAB7225) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to nuclei. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Bromodeoxyuridine/BrdU in Human Breast Cancer Tissue. Bromodeoxyuridine/BrdU was detected in immersion fixed paraffin-embedded sections of human breast cancer tissue using Mouse Anti-Bromodeoxyuridine/BrdU Monoclonal Antibody (Catalog # MAB7225) at 1 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (VC001). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cell nuclei. Staining was performed using our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Bromodeoxyuridine/BrdU in Human Testis. Bromodeoxyuridine/BrdU was detected in immersion fixed paraffin-embedded sections of human testis using Mouse Anti-Bromodeoxyuridine/BrdU Monoclonal Antibody (Catalog # MAB7225) at 1 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (VC001). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cell nuclei. Staining was performed using our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Detection of Bromodeoxyuridine/BrdU in Human PBMCs by Flow Cytometry Human peripheral blood mononuclear cells (PBMCs) were treated overnight with 50 ng/mL PMA, 500 ng/mL Ionomycin, and 30 µg/mL BrdU, then stained with Mouse Anti-Bromodeoxyuridine/BrdU Monoclonal Antibody (Catalog # MAB7225, filled histogram) or isotype control antibody (Catalog # MAB003, open histo-gram), followed by Allophycocyanin-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0101B). To facilitate intracellular staining, cells were fixed with cold, 70% ethanol for 5 minutes, DNA was denatured with 1.5M HCl for 30 minutes, and then cells were permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (Catalog # FC005).

Detection of Bromodeoxyuridine/BrdU by Immunohistochemistry SIGIRR delta E8 interacts with a mitochondrial protein ATP5A1. (A) Engineered HT-29 cells inducibly expressing SIGIRR delta E8 were incubated with 10 μM of BrdU for 6 h followed by fixation with PFA and immunofluoresence staining with anti-BrdU antibody. (B) HA pull-down experiment was performed from cells expressing a HA-tagged SIGIRR. These proteins were fractionated on an SDS-Page gel, the fractionated proteins were digested with trypsin, and the digests were analyzed by LC-MS/MS analysis. The protein, ATP5A1, was identified in the SIGIRR pull-down experiments by a total of 6 peptides (top). One of these peptides corresponds to the (134)TGAIVDVPVGEELLGR(149) peptide, which was identified as a doubly charged peptide with a mass of 812.950 Da. The CID spectra for this peptide are given above and are dominated by singly charged C-terminal y ions and N-terminal b ions (bottom). (C) HeLa cells were cotransfected with myc-tagged ATP5A1 and M2 flag-tagged SIGIRRFL and SIGIRR delta E8. Transfected cells were lysed, and the lysates were immunoprecipitated with anti-FLAG antibody followed by Western blot analysis. (D) SIGIRR protein was immunoprecipitated from indicated cell lines. Coimmunoprecipitated proteins were resolved on SDS-PAGE followed by Western blot for ATP5A1 and SIGIRR. The experiments (except MS) were repeated thrice and yielded consistent results; the representative results are shown. Data are shown as mean ± SEM. ****P < 0.0001 by 2-tailed t-test. Scale bar, 50 μm. CID, collision-induced dissociation; LC-MS/MS, liquid chromatography–mass spectrometry/mass spectrometry; PFA, paraformaldehyde; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35900274), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Bromodeoxyuridine/BrdU by Immunohistochemistry SIGIRR delta E8 interacts with a mitochondrial protein ATP5A1. (A) Engineered HT-29 cells inducibly expressing SIGIRR delta E8 were incubated with 10 μM of BrdU for 6 h followed by fixation with PFA and immunofluoresence staining with anti-BrdU antibody. (B) HA pull-down experiment was performed from cells expressing a HA-tagged SIGIRR. These proteins were fractionated on an SDS-Page gel, the fractionated proteins were digested with trypsin, and the digests were analyzed by LC-MS/MS analysis. The protein, ATP5A1, was identified in the SIGIRR pull-down experiments by a total of 6 peptides (top). One of these peptides corresponds to the (134)TGAIVDVPVGEELLGR(149) peptide, which was identified as a doubly charged peptide with a mass of 812.950 Da. The CID spectra for this peptide are given above and are dominated by singly charged C-terminal y ions and N-terminal b ions (bottom). (C) HeLa cells were cotransfected with myc-tagged ATP5A1 and M2 flag-tagged SIGIRRFL and SIGIRR delta E8. Transfected cells were lysed, and the lysates were immunoprecipitated with anti-FLAG antibody followed by Western blot analysis. (D) SIGIRR protein was immunoprecipitated from indicated cell lines. Coimmunoprecipitated proteins were resolved on SDS-PAGE followed by Western blot for ATP5A1 and SIGIRR. The experiments (except MS) were repeated thrice and yielded consistent results; the representative results are shown. Data are shown as mean ± SEM. ****P < 0.0001 by 2-tailed t-test. Scale bar, 50 μm. CID, collision-induced dissociation; LC-MS/MS, liquid chromatography–mass spectrometry/mass spectrometry; PFA, paraformaldehyde; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35900274), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Bromodeoxyuridine/BrdU by Immunocytochemistry/ Immunofluorescence Disruption of the TAGLN2-YBX1-AKT interaction reduces cytosolic ssDNA accumulation, ISGs expression, and drug resistance.A~C Multiplex immunofluorescence of TMA was performed using the Opal 7-color Manual IHC Kit and VECTASHIELD® HardSet Antifade Mounting Medium. The multiplex antibody panel was optimized as follows: TAGLN2, Opal 520 (yellow); CK, Opal 570 (green); YBX1, Opal 620 (red). The TMA was counterstained with DAPI (blue) and incubated with an antifluorescence quencher. Expression and spatial distribution of TAGLN2 or YBX1 in tissues and the correlation with patient clinical data. The DAPI channel was used to identify individual cells. A tissue segmentation algorithm combined with CK staining was applied to define tumoral and stromal areas. The scale bar is 200 μm. D Fisetin or MK2206 inhibited the accumulation of cytosolic ssDNA induced by overexpression of TAGLN2 by BrdU-gamma H2AX double labeling. HGC-27 cells stably transfected with TAGLN2 were prelabeled with BrdU and subsequently treated with 1 μg/ml Cisplatin with or without 10 μM Fisetin or 200 nM MK2206 in the medium. Cells were stained for DNA (DAPI, blue), the primary BrdU antibody (red) and phospho-histone H2AX (green). E Relative mRNA levels of the panel of IFN-related genes with or without 10 μM Fisetin or 200 nM MK2206 in the medium after 6 Gy X-ray treatment were evaluated by quantitative RT‒PCR analysis. The cytotoxicity induced by MK2206 from 16.25 nM to 13 μM (F) or MK2206 (200 nM) and Cisplatin (0.4 μg/ml) combination on tumor cells was detected (G). *P < 0.05, **P < 0.01, ***P < 0.001. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/39168971), licensed under a CC-BY license. Not internally tested by R&D Systems.