6 In Situ Hybridization for DNA: Fluorescent Probe
77
Slides containing cells are prepared using methanol-acetic acid Remarks
(3:1) fixation.
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.
For single copy hybridization, the slides are treated with
RNAse, Proteinase K, and paraformaldehyde. Apply 200 fll of
RNAse (100 flg/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 of2XSSC 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 /PBS 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 - 6flg/ml in 20 mM Tris/2 mM CaCh pH 7.5.
1. Immerse the slides in 2 x SSC for 30mins at 37°C in order to Slide prepara"age" them, if specimens were freshly prepared within the last tion for cell
24 hrs. Dehydrate the slides through a series of diluted etha- specimen
nol. Air dry.
2. 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 hybridizaiton area might be 20 mm square. The hybridization
and multiple cytochemical reactions are carried out under
coverslips to minimize the amount of reagents required.
3. 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.
4. Transfer the slide quickly to 70% ethanol for 1 minute. Agitate
to rinse off denaturing solution.
5. Continue dehydration in 85% ethanol for 1 minute, and
1 minute in 100% ethanol.
77
Slides containing cells are prepared using methanol-acetic acid Remarks
(3:1) fixation.
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.
For single copy hybridization, the slides are treated with
RNAse, Proteinase K, and paraformaldehyde. Apply 200 fll of
RNAse (100 flg/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 of2XSSC 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 /PBS 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 - 6flg/ml in 20 mM Tris/2 mM CaCh pH 7.5.
1. Immerse the slides in 2 x SSC for 30mins at 37°C in order to Slide prepara"age" them, if specimens were freshly prepared within the last tion for cell
24 hrs. Dehydrate the slides through a series of diluted etha- specimen
nol. Air dry.
2. 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 hybridizaiton area might be 20 mm square. The hybridization
and multiple cytochemical reactions are carried out under
coverslips to minimize the amount of reagents required.
3. 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.
4. Transfer the slide quickly to 70% ethanol for 1 minute. Agitate
to rinse off denaturing solution.
5. Continue dehydration in 85% ethanol for 1 minute, and
1 minute in 100% ethanol.