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ApoBrdU DNA Fragmentation Assay Kit

ABIN411728 产品详细信息, 供应商: Log in to see
适用
DNA
应用范围
Detection (D), Fluorescence Microscopy (FM), Flow Cytometry (FACS)
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原理 Apo-BrdU In Situ DNA Fragmentation Assay Kit provides complete components including positive and negative control cells for conveniently detecting DNA fragmentation by fluorescence microscopy or flow cytometry. The kit utilizes Br-dUTP (bromolated deoxyuridine triphosphate nucleotides) which is more readily incorporated into DNA strand breaks than other larger ligands (e.g., fluorescein, biotin or digoxigenin). The greater incorporation gives rise to brighter signal when the Br-dUTP sites are identified by a fluorescein labeled anti-BrdU monoclonal antibody.
品牌 ApoBrdU™
样品类型 Adherent Cell Culture, Cell Culture Cells, Biological Fluids, Tissue Samples
检测方法 Fluorometric
产品特性 Apo-BrdU In Situ DNA Fragmentation Assay Kit: Convenient & Reliable Assay to Detect DNA Fragmentation by Fluorescence Microscopy or Flow Cytometry. Includes both Positive and Negative Control Cells.
组件
  • Positive Control Cells
  • Negative Control Cells
  • Wash Buffer
  • Reaction Buffer
  • TdT Enzymes
  • Br-dUTP
  • Rinse Buffer
  • Anti-BrdU-FITC Antibody
  • PI/RNase Staining Buffer
背景 Internucleosomal DNA fragmentation is a hallmark of apoptosis in mammalian cells.
应用备注 Detection method: Flow cytometry (Ex/Em = 488/520 nm for FITC, and 488/623 nm for PI) and Fluorescence microscopy (FITC and rhodamine filters)
Applications: The TUNEL-based assay kit provides complete components including positive and negative control cells for convenient detection of DNA fragmentation in cultured cells and tissue sections.
说明

Flow cytometry (Ex/Em = 488/520 nm for FITC, and 488/623 nm for PI) and fluorescence microscopy (FITC and rhodamine filters)
Simple procedure, takes ~ 1 hour
Fast and convenient
The assay is sensitive and stable
The incorporation of BrdU in the procedure provides greater sensitivity than biotinylated or digoxigenylated methods.

实验流程 Note: If using fresh-frozen tissue sections, proceed directly to
7. 1. Remove paraffin by immersing slides in a Coplin jar containing fresh xylene. Incubate at room temperature for 5 minutes. 2. Repeat in a second Coplin jar containing fresh xylene. 3. Immerse the slides in a Coplin Jar containing 100 % ethanol and incubate at room temperature for 5 min.
4. Rehydrate the slides by sequential 3-min, room temperature incubations in Coplin jars containing:
100 % ethanol
95 % ethanol
85 % ethanol
70 % ethanol
50 % ethanol
5. Immerse the slides in a Coplin jar containing 0.85 % NaCl and incubate at room temperature for 5 min.
6. Immerse the slides in a Coplin jar containing PBS and incubate at room temperature for 5 minutes.
7. fix the slides by immersing them in a Coplin jar containing fresh 4 % formaldehyde/PBS, and incubate at room temperature for 15 min.
8. Wash the slides by immersing them in a Coplin jar containing PBS, and incubate at room temp. for 5 min.
9. Transfer to another Coplin jar containing PBS, and incubate at room temperature for 5 min. 10. Allow the liquid to drain thoroughly and place slides on a flat surface. 11. Prepare 20 µg/mL of Proteinase K Solution (combine 2 µL of 10 mg/mL Protease K and 998 µL of 100 mM Tris-HCl, pH 8.0, 50 mM EDTA) and cover each section with 100 µL of it. Incubate at room temperature for 5 min. 12. Immerse the slides in Coplin jar containing PBS, and incubate at room temperature for 5 min. 13. Transfer the slides to a Coplin jar containing 4 % formaldehyde/PBS and incubate at room temperature for 5 minutes. 14. Wash the slides by immersion in Coplin jar containing PBS, and incubate at room temperature for 5 min. B. Detection by Fluorescence Microscopy: 1. Remove slides from PBS and tap gently to remove excess liquid. Cover the cells in 100 ul of Wash buffer (blue cap). 2. Use forceps, gently place a piece of plastic coverslip on top of the cells to evenly spread the liquid, incubate for 5 min. Remove plastic coverslip and gently tap the slides to remove excess liquid. 3. Repeat step 2. Carefully blot dry around the edges with tissue paper.
4. Gently place 50 µL of the DNA Labeling Solution (prepared as in Section IIIB, Step 4) on the cells.
5. Use forceps, gently place a piece of plastic coverslip on top of the cells to evenly spread the liquid.
6. Place the slides in a dark, humidified 37 °C incubator for 60 minutes. Ensure high humidity by placing wet paper towels in the bottom of the dry incubator.
7. Using forceps, remove the plastic coverslips. Rinse the slides to a fresh Coplin jar filled with PBS for 5 min.
8. Repeat
7. Carefully blot dry around the edges with tissue paper.
9. Place 100 µL of the Antibody Solution (Prepared as in Section IIIB, step 8). Antibody Solution 1 assay 10 assays Anti-BrdU-FITC Antibody (orange cap) 5 µL 50 µL Rinse Buffer (red cap) 95 µL 950 µL The Kit rev. 10/07
限制 仅限研究用
储存条件 -20 °C
有效期 12 months
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