74
MORIMASA MATS UTA AND MAYUMI MATSUTA
For single copy hybridization, the slides are treated with
RNAse, Proteinase K, and paraformaldehyde. Apply 200 /ll of
RNAse (100 /lg/ml in 2XSSC) to each slide and cover with a large
plastic coverslip (24 X 50 ~ 60 mm). Place slides in a moist chamber
at 37°C for one hour. Rinse in four changes of 2XSSC at room
temperature, dehydrate in an ethanol series and air dry. The slides
are then treated with Proteinase K for 7.5 mins at 37°C, washed in
2XSSC, fixed in 4% paraformaldehyde in phosphate buffer (PB)
for 10 mins, and washed in 2XSSC. The Proteinase K concentration
is high enough so that without the fixation the chromosomes
would be destroyed in denaturation procedure, suggesting concentration at 0.6 - 6/lg/ml in 20 mM Tris/2 mM CaClz pH 7.5.
Slide Note: Immerse the slides in 2XSSC for 30 mins at 37°C in order to
preparation "age" them, if the specimen were freshly prepared within the last
24 hours. Dehydrate the slides through a series of 1 minute ethanol washes (70%, 85%, 100%). Air dry. This may help chromosomes maintain morphology during the hybridization.
1. Locate best area on slide using low power microscopy. Mark
the back of the slide with a diamond scribe to indicate roughly
where the boundaries of the coverslip should be. A typical hybridization area might be a square of 20 mm. The hybridization and multiple cytochemical reactions are carried out under coverslips to minimize the amount of reagents required.
2. Immerse the slides in the 74°C denaturation solution (70%
formamidel2XSSC) for 5 mins.
Note: A room temperature slide put into 50 ml Coplin jar will
cause the temperature to drop by about 1°C, so do only a few
slides at a time.
3. Transfer the slide quickly to 70% ethanol for 1 minute. Agitate
to rinse off denaturing solution.
4. Continue dehydration in 85% ethanol for 1 minute, and
1 minute in 100% ethanol.
5. Remove the slides from the 100% ethanol.
6. Dry slides by touching the bottom edge of the slides to a blotter and wiping the underside of the slides dry with a paper
towel.
MORIMASA MATS UTA AND MAYUMI MATSUTA
For single copy hybridization, the slides are treated with
RNAse, Proteinase K, and paraformaldehyde. Apply 200 /ll of
RNAse (100 /lg/ml in 2XSSC) to each slide and cover with a large
plastic coverslip (24 X 50 ~ 60 mm). Place slides in a moist chamber
at 37°C for one hour. Rinse in four changes of 2XSSC at room
temperature, dehydrate in an ethanol series and air dry. The slides
are then treated with Proteinase K for 7.5 mins at 37°C, washed in
2XSSC, fixed in 4% paraformaldehyde in phosphate buffer (PB)
for 10 mins, and washed in 2XSSC. The Proteinase K concentration
is high enough so that without the fixation the chromosomes
would be destroyed in denaturation procedure, suggesting concentration at 0.6 - 6/lg/ml in 20 mM Tris/2 mM CaClz pH 7.5.
Slide Note: Immerse the slides in 2XSSC for 30 mins at 37°C in order to
preparation "age" them, if the specimen were freshly prepared within the last
24 hours. Dehydrate the slides through a series of 1 minute ethanol washes (70%, 85%, 100%). Air dry. This may help chromosomes maintain morphology during the hybridization.
1. Locate best area on slide using low power microscopy. Mark
the back of the slide with a diamond scribe to indicate roughly
where the boundaries of the coverslip should be. A typical hybridization area might be a square of 20 mm. The hybridization and multiple cytochemical reactions are carried out under coverslips to minimize the amount of reagents required.
2. Immerse the slides in the 74°C denaturation solution (70%
formamidel2XSSC) for 5 mins.
Note: A room temperature slide put into 50 ml Coplin jar will
cause the temperature to drop by about 1°C, so do only a few
slides at a time.
3. Transfer the slide quickly to 70% ethanol for 1 minute. Agitate
to rinse off denaturing solution.
4. Continue dehydration in 85% ethanol for 1 minute, and
1 minute in 100% ethanol.
5. Remove the slides from the 100% ethanol.
6. Dry slides by touching the bottom edge of the slides to a blotter and wiping the underside of the slides dry with a paper
towel.