6 In Situ Hybridization for DNA: Fluorescent Probe
79
17. Place slides in a prewarmed humidified box and place box in
a 37°C incubator for 6-16 hours.
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 Washing the
for washing, and a jar of 2X sse. Place the jar in a 46°C water slide
bath 30 mins prior to use in order to attain the solution temperature.
18. 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.
19. Immerse slides in jar #2 of wash solution at 46°e. Agitate
slides for 1-3 sees. Remove slides after 10 mins.
20. Immerse slides in jar #3 of wash solution at 46°e. Agitate
slides for 1-3 sees. Remove slides after 10 mins.
21. Immerse slides in 2XSSC at 46°C. Agitate slides for 1-3 sees.
Remove slides after 10 mins.
22. Immerse slides in PN buffer at room temperature for 1 minute.
Note: You may apply the rapid wash procedure in Protocol for
Rapid Procedure (Subprotocol 2).
Note: The slide should not be allowed to dry because the resulting salt crystals cause nonspecific binding of the detection reagents.
23. Remove a slide from PN buffer, drain and blot excess liquid
form edge. Apply diluted avidin-FITC or -Texas-Red (5 ).1l!
ml in PNM buffer, 5).1l/cm2), and cover with a plastic coverslip cut from a polyethylene bag.
Fluorescent
staining using
biotinized
probes
79
17. Place slides in a prewarmed humidified box and place box in
a 37°C incubator for 6-16 hours.
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 Washing the
for washing, and a jar of 2X sse. Place the jar in a 46°C water slide
bath 30 mins prior to use in order to attain the solution temperature.
18. 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.
19. Immerse slides in jar #2 of wash solution at 46°e. Agitate
slides for 1-3 sees. Remove slides after 10 mins.
20. Immerse slides in jar #3 of wash solution at 46°e. Agitate
slides for 1-3 sees. Remove slides after 10 mins.
21. Immerse slides in 2XSSC at 46°C. Agitate slides for 1-3 sees.
Remove slides after 10 mins.
22. Immerse slides in PN buffer at room temperature for 1 minute.
Note: You may apply the rapid wash procedure in Protocol for
Rapid Procedure (Subprotocol 2).
Note: The slide should not be allowed to dry because the resulting salt crystals cause nonspecific binding of the detection reagents.
23. Remove a slide from PN buffer, drain and blot excess liquid
form edge. Apply diluted avidin-FITC or -Texas-Red (5 ).1l!
ml in PNM buffer, 5).1l/cm2), and cover with a plastic coverslip cut from a polyethylene bag.
Fluorescent
staining using
biotinized
probes