80
MORIMASA MATS UTA AND MAYUMI MATS UTA
Amplification
of the biotinized probe
fluorescence
24. The slide is then incubated for 20 mins in a moist chamber at
room temperature in darkness.
Note: The amount of avidin used here is in excess so dilution as it
is applied to the wet slide is not critical. The concentration is
sufficient even for the amplification steps, where there are
many times more binding sites available.
25. Wash the slides in 3 changes ofPN buffer at room temperature for 2 mins each.
26. Mount in an anti-fade solution (l.5 J.ll/cm 2 ) containing DAP,
or PI for FITC, and a no. 1 thickness coverslip used. View
using fluorescent microscope.
Note: Amplification of the biotinized probe fluorescence is accomplished by applying a biotinylated goat -anti-avidin antibody
followed by another layer of avidin after Step 25. This can be
done even after observation of the slide after Step 26, followed
by removing cover slips and washes as in Step 25 in PN buffer.
1. Drain and blot the excess liquid from the slide.
2. Add the biotinylated goat-anti-avidin antibody solution
(100 times diluted in PNM buffer), and incubate at room
temperature for 20 mins under a plastic coverslip.
3. Wash the slides in 3 changes ofPN buffer at room temperature for 2 mins each.
4. Remove a slide from PN buffer, drain and blot excess liquid
form edge.
5. Apply diluted avidin-FITC or -Texas-Red (5J.ll/ml in PNM
buffer, 5J.ll/cm 2 ), and cover with a plastic coverslip.
6. The slide is then incubated for 20 mins in a moist chamber at
room temperature in darkness.
7. Wash the slides in 3 changes ofPN buffer at room temperature for 2 mins each.
8. Mount in an anti-fade solution (l.5 J.lI/cm 2 ) containing DAPI
or propidium iodide (PI) for FITC, and a no. 1 thickness coverslip used. View using fluorescent microscope.
MORIMASA MATS UTA AND MAYUMI MATS UTA
Amplification
of the biotinized probe
fluorescence
24. The slide is then incubated for 20 mins in a moist chamber at
room temperature in darkness.
Note: The amount of avidin used here is in excess so dilution as it
is applied to the wet slide is not critical. The concentration is
sufficient even for the amplification steps, where there are
many times more binding sites available.
25. Wash the slides in 3 changes ofPN buffer at room temperature for 2 mins each.
26. Mount in an anti-fade solution (l.5 J.ll/cm 2 ) containing DAP,
or PI for FITC, and a no. 1 thickness coverslip used. View
using fluorescent microscope.
Note: Amplification of the biotinized probe fluorescence is accomplished by applying a biotinylated goat -anti-avidin antibody
followed by another layer of avidin after Step 25. This can be
done even after observation of the slide after Step 26, followed
by removing cover slips and washes as in Step 25 in PN buffer.
1. Drain and blot the excess liquid from the slide.
2. Add the biotinylated goat-anti-avidin antibody solution
(100 times diluted in PNM buffer), and incubate at room
temperature for 20 mins under a plastic coverslip.
3. Wash the slides in 3 changes ofPN buffer at room temperature for 2 mins each.
4. Remove a slide from PN buffer, drain and blot excess liquid
form edge.
5. Apply diluted avidin-FITC or -Texas-Red (5J.ll/ml in PNM
buffer, 5J.ll/cm 2 ), and cover with a plastic coverslip.
6. The slide is then incubated for 20 mins in a moist chamber at
room temperature in darkness.
7. Wash the slides in 3 changes ofPN buffer at room temperature for 2 mins each.
8. Mount in an anti-fade solution (l.5 J.lI/cm 2 ) containing DAPI
or propidium iodide (PI) for FITC, and a no. 1 thickness coverslip used. View using fluorescent microscope.