6 In Situ Hybridization for DNA: Fluorescent Probe
89
- 2)11 probe (fluorochrome-directly-labeled :diluted if necessary)
- total 10)11, for each target area (I8mmXI8mm)
2. Centrifuge tube for 1-3 sees.
3. Vortex and re-centrifuge.
4. Place tube in a 74°C water bath for 5 mins.
5. Remove tube from water bath.
6. Place tube on a slide warmer set to 45-50°C.
7. Mark hybridization areas with a diamond tipped scribe on Slide
the specimen slides.
preparation
8. Immerse the slides in the 74°C denaturation solution (70%
formamide12XSSe) for 5 mins.
9. Dehydrate slides 1 minute in 70% ethanol, 1 minute in 85%
ethanol, and 1 minute in 100% ethanol.
10. Remove the slides from the 100% ethanol.
11. 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.
12. Place slides on a 45-50 o e slide warmer to evaporate remaining ethanol for up to 2 mins.
13. Apply 10 1)11 of hybridization mix to slide. Apply coverslip Hybridization
on placing hybridization mix.
Ifhybridization time will be greater than 2 hours, seal coverslip with rubber cement.
14. Place slides in a prewarmed non-humidified box and place
box in a 42°e incubator for 30 mins -60 mins.
15. Remove the coverslip and immediately immerse the slide in
jar ofO.4X sse wash solution at 74°C. Agitate slide for 1-3
sees. Remove slide after 2 mins. If many slides were hybridized, do not remove the coverslips from several slides before placing any of the slides in the wash solution. Begin timing the incubation when the last slide has been added to the
wash solution.
Washing the
slide
89
- 2)11 probe (fluorochrome-directly-labeled :diluted if necessary)
- total 10)11, for each target area (I8mmXI8mm)
2. Centrifuge tube for 1-3 sees.
3. Vortex and re-centrifuge.
4. Place tube in a 74°C water bath for 5 mins.
5. Remove tube from water bath.
6. Place tube on a slide warmer set to 45-50°C.
7. Mark hybridization areas with a diamond tipped scribe on Slide
the specimen slides.
preparation
8. Immerse the slides in the 74°C denaturation solution (70%
formamide12XSSe) for 5 mins.
9. Dehydrate slides 1 minute in 70% ethanol, 1 minute in 85%
ethanol, and 1 minute in 100% ethanol.
10. Remove the slides from the 100% ethanol.
11. 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.
12. Place slides on a 45-50 o e slide warmer to evaporate remaining ethanol for up to 2 mins.
13. Apply 10 1)11 of hybridization mix to slide. Apply coverslip Hybridization
on placing hybridization mix.
Ifhybridization time will be greater than 2 hours, seal coverslip with rubber cement.
14. Place slides in a prewarmed non-humidified box and place
box in a 42°e incubator for 30 mins -60 mins.
15. Remove the coverslip and immediately immerse the slide in
jar ofO.4X sse wash solution at 74°C. Agitate slide for 1-3
sees. Remove slide after 2 mins. If many slides were hybridized, do not remove the coverslips from several slides before placing any of the slides in the wash solution. Begin timing the incubation when the last slide has been added to the
wash solution.
Washing the
slide