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HARALD RIEDER et al.
24. Place a 22 x 22 mm coverslip on the hybridization area, seal with rubber
cement, dry on heating plate.
25. Place slides in a humidified box and incubate in a 3TC incubator overnight (at least 14-18 h).
Third day: post-hybridization procedure
Prewarm Coplin jar containing 2x SSC/O.3% NP 40 in a 73°C water bath.
Washing 1. Carefully remove rubber cement with forceps.
2. Immerse slides in 2x SSC/O.3% NP 40 at room temperature and float
off coverslips.
3. Place slides into 2xSSC/0.3% NP 40 at 73°C: 2 min.
Note: Make sure that the temperature has reached 71-73°C after adding
all slides, at most 6 slides/jar.
More rapidly, the solution can be prewarmed to 73°C by a microwave if the
temperature in the Coplin jar is controlled by a sensor. When proceeding
immediately, the washing procedure may even be performed without
using a water bath because the decrease in the temperature during incubation is negligible.
4. Place slides into 2x SSC/0.3% NP40 at room temperature.
5. Immerse slides in 2x SSC: 5 min.
6. Immerse slides in DAPI/2xSSC: 5 min.
7. Immerse slides in 2xSSC: 5 min.
8. Remove excess liquid by wicking off the edge of the slide, add one drop
of mounting medium (e.g.Vectashield)/slide and cover with a 22x60
mm coverslip.
9. Analyze hybridization signals using an epifluorescence microscope
equipped with filter combinations adequate for the fluorochromes
used.
Analysis (basic remarks for signal evaluation in tissue sections): To provide reliable results, the counting of overlapping and ruptured nuclei
should be avoided. Out of focus signals should be considered and focus-
HARALD RIEDER et al.
24. Place a 22 x 22 mm coverslip on the hybridization area, seal with rubber
cement, dry on heating plate.
25. Place slides in a humidified box and incubate in a 3TC incubator overnight (at least 14-18 h).
Third day: post-hybridization procedure
Prewarm Coplin jar containing 2x SSC/O.3% NP 40 in a 73°C water bath.
Washing 1. Carefully remove rubber cement with forceps.
2. Immerse slides in 2x SSC/O.3% NP 40 at room temperature and float
off coverslips.
3. Place slides into 2xSSC/0.3% NP 40 at 73°C: 2 min.
Note: Make sure that the temperature has reached 71-73°C after adding
all slides, at most 6 slides/jar.
More rapidly, the solution can be prewarmed to 73°C by a microwave if the
temperature in the Coplin jar is controlled by a sensor. When proceeding
immediately, the washing procedure may even be performed without
using a water bath because the decrease in the temperature during incubation is negligible.
4. Place slides into 2x SSC/0.3% NP40 at room temperature.
5. Immerse slides in 2x SSC: 5 min.
6. Immerse slides in DAPI/2xSSC: 5 min.
7. Immerse slides in 2xSSC: 5 min.
8. Remove excess liquid by wicking off the edge of the slide, add one drop
of mounting medium (e.g.Vectashield)/slide and cover with a 22x60
mm coverslip.
9. Analyze hybridization signals using an epifluorescence microscope
equipped with filter combinations adequate for the fluorochromes
used.
Analysis (basic remarks for signal evaluation in tissue sections): To provide reliable results, the counting of overlapping and ruptured nuclei
should be avoided. Out of focus signals should be considered and focus-