76
MORIMASA MATSUTA AND MAYUMI MATSUTA
Washing
the slide
Note: To produce an assay with sufficient signal, start with over
night hybridization for most probes. With repetitive probes the
hybridization incubation can be shortened to 30 mins (signals
are visible after 10 mins). The hybridization signal reaches a
maximum after about 6-8 hours.
Prepare 3 Coplin jars of 50% formamide/2XSSC wash solution for
washing, and a jar of2X sse. Place the jar in a 46°C water bath 30
mins prior to use in order to attain the solution temperature.
17. Remove slides from incubator and peel off rubber cement
with forceps, and immediately immerse the slides in jar
#1 of 50% formamide/2XSSC wash solution at 46°e. Agitate
slides for 1-3 sees. The coverslips can be slid off easily in the
first wash. Do this gently so the specimens are not scratched.
Remove slides after 10 mins.
18. Immerse slides in jar #2 of wash solution at 46°e. Agitate
slides for 1-3 sees. Remove slides after 10 mins.
19. Immerse slides in jar #3 of wash solution at 46°e. Agitate
slides for 1-3 sees. Remove slides after 10 mins.
20. Immerse slides in 2XSSC at 46°C. Agitate slides for 1-3 sees.
Remove slides after 10 mins.
21. Immerse slides in 2XSSC/O.l % Nonidet P-40 (NP-40) at
room temperature for 1 minute.
22. Allow slides to air dry in darkness.
23. Mount in an anti-fade solution containing DAPI or propidium iodide (PI) for green fluorochromes (FITC, SpectrumGreen, etc.). View using fluorescent microscope.
Note: You may apply the rapid wash procedure in Protocol for
Rapid Procedure.
FISH using chemically modified DNA probes
Note: If the probes are modified with haptens, such as biotin or
digoxigenin etc., fluorescent staining procedures should follow.
These probes yield brighter signals than fluorochrome-directlylabeled probes with slightly high background.
MORIMASA MATSUTA AND MAYUMI MATSUTA
Washing
the slide
Note: To produce an assay with sufficient signal, start with over
night hybridization for most probes. With repetitive probes the
hybridization incubation can be shortened to 30 mins (signals
are visible after 10 mins). The hybridization signal reaches a
maximum after about 6-8 hours.
Prepare 3 Coplin jars of 50% formamide/2XSSC wash solution for
washing, and a jar of2X sse. Place the jar in a 46°C water bath 30
mins prior to use in order to attain the solution temperature.
17. Remove slides from incubator and peel off rubber cement
with forceps, and immediately immerse the slides in jar
#1 of 50% formamide/2XSSC wash solution at 46°e. Agitate
slides for 1-3 sees. The coverslips can be slid off easily in the
first wash. Do this gently so the specimens are not scratched.
Remove slides after 10 mins.
18. Immerse slides in jar #2 of wash solution at 46°e. Agitate
slides for 1-3 sees. Remove slides after 10 mins.
19. Immerse slides in jar #3 of wash solution at 46°e. Agitate
slides for 1-3 sees. Remove slides after 10 mins.
20. Immerse slides in 2XSSC at 46°C. Agitate slides for 1-3 sees.
Remove slides after 10 mins.
21. Immerse slides in 2XSSC/O.l % Nonidet P-40 (NP-40) at
room temperature for 1 minute.
22. Allow slides to air dry in darkness.
23. Mount in an anti-fade solution containing DAPI or propidium iodide (PI) for green fluorochromes (FITC, SpectrumGreen, etc.). View using fluorescent microscope.
Note: You may apply the rapid wash procedure in Protocol for
Rapid Procedure.
FISH using chemically modified DNA probes
Note: If the probes are modified with haptens, such as biotin or
digoxigenin etc., fluorescent staining procedures should follow.
These probes yield brighter signals than fluorochrome-directlylabeled probes with slightly high background.