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Applikationshinweise
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Note: If using fresh-frozen tissue sections, proceed directly to step 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 μ g/ml of Proteinase K Solution (combine 2 μ l of 10 mg/ml Protease K and 998 μ l of 100 mM Tris-HCl, pH 8.0, 50 mM EDTA) and cover each section with 100 μ l 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 μ l 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 o 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 step 7. Carefully blot dry around the edges with tissue paper. 9. Place 100 μ l 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 BioVision rev. 10/07
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