IN SITU HYBRIDIZATION: (Day 3)

Post-hybridization wash:

1. Prewarm post-wash solution (60% formamide, 2X SSC) to 37℃ in a coplin jar. In addition, prewarm 100 ml of 2X SSC to 37℃ (50 ml in each of two coplin jars).
2. Remove slides from humid chamber, peel off coverslips and incubate slides 20 minutes in the 37℃ post-wash solution (agitate slides every 5 minutes).
3. Transfer slides to the first coplin jar containing 2X SSC. Incubate slides for 5 minutes at 37℃ (agitate slides after 2 minutes). Transfer slides to the second SSC containing coplin jar and incubate as described for the first SSC coplin jar.
4. Transfer slides to a coplin jar containing room temperature 1X PBD (phosphate buffered detergent). Incubate for 2 minutes (agitate slides after 1 minute).
   DO NOT ALLOW WELLS TO DRY AT ANY TIME PAST THIS POINT!!!!!!
   
   Detection: This protocol can be used to detect either digoxigenin or biotinylated DNA probes. We use digoxigenin probes, primarily because it is much cheaper.
   a) Digoxigenin probes: Detection by i) mouse anti-DIG antibody, ii) FITC conjugated goat anti-mouse antibody and iii) FITC conjugated pig anti-goat antibody. All antibodies are diluted 1:250 in 10% horse serum just prior to use.
   b)Biotinylated probes: Detection is via FITC-avidin and anti-avidin antibodies (from the ONCOR in situ hybridization kit).
   Note: preparation of digoxigenin or biotin labelled probes is described in equipment and solutions section.
5. Remove one slide at a time from 1X PBD, quickly tilt slide to drain off excess liquid and blot off liquid near slide edges. Digoxigenin probes: Immediately add 7.5 ul 10% horse serum to each well containing a hybridized probe. Incubate 5 minutes at room temperature in a humid chamber. Add 7.5 ul Mouse anti-DIG antibody to each well. Incubate 20 minutes at 37℃ in a humid chamber (prewarmed to 37℃). Biotinylated probes: follow digoxigenin probe instructions except use 7.5 ul blocking reagent 1 from ONCOR kit instead of horse serum then add 7.5 ul FITC-avidin to each well instead of mouse antibody.
   
   Amplification:
6. Remove slides from humid chamber and incubate slides for 2 minutes in a coplin jar containing 1X PBD (room temperature). Agitate slides after one minute. Repeat wash 2X in fresh 1X PBD at room temperature for 2 minutes/wash.
7. Remove one slide at a time from 1X PBD, quickly tilt slide to drain off excess liquid and blot off excess liquid near wells. Digoxigenin probes: Immediately add 7.5 ul 10% horse serum to each well containing a hybridized probe. Incubate 5 minutes at room temperature in a humid chamber. Add 7.5 ul FITC conjugated goat anti-mouse antibody to each well. Incubate 20 minutes at 37℃ in a humid chamber (prewarmed to 37℃). Biotinylated probes: follow digoxigenin probe instructions except use 7.5 ul blocking reagent 2 from ONCOR kit instead of horse serum then add 7.5 ul anti-avidin antibodies to each well instead of goat antibody.
8. Repeat PBD washes as described in step 26.
9. Remove one slide at a time from 1X PBD, quickly tilt slide to drain off excess liquid and blot off excess liquid near wells. Digoxigenin probes: Immediately add 7.5 ul 10% horse serum to each well containing a hybridized probe. Incubate 5 minutes at room temperature in a humid chamber. Add 7.5 ul FITC conjugated pig anti-goat antibody to each well. Incubate 20 minutes at 37℃ in a humid chamber (prewarmed to 37℃). Biotinylated probes: follow digoxigenin probe instructions except use 7.5 ul blocking reagent 1 from ONCOR kit instead of horse serum then add 7.5 ul FITC-avidin instead of pig antibody.
   
   Chromosome staining:
10. Repeat PBD washes as described in step 26.
11. Remove one slide at a time from 1X PBD, tilt slide to drain off excess liquid and blot off excess liquid near wells.
12. Add 5 ul propidium iodide/antifade solution to each well (warm to room temperature before adding since this solution is very viscous at -20℃). Cover with glass coverslip. Remove any bubbles by pressing coverslip gently with the eraser end of a pencil.
13. Fill a 1 cc syringe with rubber cement and seal the coverslip to the slide by adding rubber cement along the coverslip edges.
14. View using fluorescence microscope. (store slides at -20℃).

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