90
MORIMASA MATS UTA AND MAYUMI MATSUTA
Preparation of
a slide for (0denaturation
16. Wash slides in 2XSSC/O.1% NP-40 at room temperature for
1 minute.
17. Allow slide to air dry in darkness.
18. Mount in an anti-fade solution containing DAPI or propidium iodide (PI). View using a fluorescent microscope.
Subprotocol 3
Protocol for Codenaturation
Procedure
Co denaturation is a process that simplifies FISH by combining of
hybridization mix and specimen into a single step. Codenaturations are performed by placing the specimen slides with probes
and coverslips applied on the surface of a hot plate that is at the
denaturation temperature. After 5 mins, the slides are removed
and are incubated at the hybridization temperature. Conditions
specify a broad range of temperatures and time, reflecting the
need to optimize conditions for specific applications and specimen types.
Note: Heat or enzymatic treatment may induce loss of adherence
of cells and tissue sections to glass slides. This may be overcome
by using aminoalkylsilane-coated glass slides.
1. Add the following to a micro centrifuge tube at ambient temperature:
- 7)11 of master mix
- 1)11 carrier DNA (500)1g/ml)
- 2)11 probe (fluorochrome-directly-labeled or haptenlabeled: diluted if necessary) total 10 )11, for each target
area (I8mmXI8mm)
2. Centrifuge tube for 1-3 secs.
3. Vortex and re-centrifuge.
4. Mark hybridization areas with a diamond tipped pen on the
specimen slide.
MORIMASA MATS UTA AND MAYUMI MATSUTA
Preparation of
a slide for (0denaturation
16. Wash slides in 2XSSC/O.1% NP-40 at room temperature for
1 minute.
17. Allow slide to air dry in darkness.
18. Mount in an anti-fade solution containing DAPI or propidium iodide (PI). View using a fluorescent microscope.
Subprotocol 3
Protocol for Codenaturation
Procedure
Co denaturation is a process that simplifies FISH by combining of
hybridization mix and specimen into a single step. Codenaturations are performed by placing the specimen slides with probes
and coverslips applied on the surface of a hot plate that is at the
denaturation temperature. After 5 mins, the slides are removed
and are incubated at the hybridization temperature. Conditions
specify a broad range of temperatures and time, reflecting the
need to optimize conditions for specific applications and specimen types.
Note: Heat or enzymatic treatment may induce loss of adherence
of cells and tissue sections to glass slides. This may be overcome
by using aminoalkylsilane-coated glass slides.
1. Add the following to a micro centrifuge tube at ambient temperature:
- 7)11 of master mix
- 1)11 carrier DNA (500)1g/ml)
- 2)11 probe (fluorochrome-directly-labeled or haptenlabeled: diluted if necessary) total 10 )11, for each target
area (I8mmXI8mm)
2. Centrifuge tube for 1-3 secs.
3. Vortex and re-centrifuge.
4. Mark hybridization areas with a diamond tipped pen on the
specimen slide.