6 In Situ Hybridization for DNA: Fluorescent Probe
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- Fluorescein-avidin (Vector Laboratories, A-20ll)
- Biotinylated Anti-avidin (Vector Laboratories, BA-0300)
- Texas-Red-avidin (Vector Laboratories, A-2016)
- Anti-digoxigen-fluorescein, Fab fragments (Boehringer
Mannheim, 1207 741)
- Anti-digoxigen-rhodamine, Fab fragments (Boehringer
Mannheim, 1207 750)
- Aminoalkylsilane (APES) coated glass slides:
- 1 ml of 3-aminopropylriethoxysilane in 50 ml acetone.
- Slides should be dipped into this solution for 20 sees, then
washed twice in acetone and twice in distilled water. Dry
slides at 37°C overnight.
- Denaturation solution: 50 ml of 70% formamide/2X SSC
- 35 ml formamide
- 5ml20X SSC
- 7 ml purified water
- Adjust pH to 7.0 with IN HCI, adjust final volume to 50 ml
with purified water. This solution can be reused for a few
weeks as long as stored at 4°C.
- Washing solution: 150 ml of 50% fromamidel2X SSC
- 75ml formamide
- 15 ml20X SSC
- 60 ml purified water
- Adjust pH to 7.0 with IN HCl. This solution can be reused
for a few weeks as long as stored at 4°C.
- Washing solution for sub-protocol 2: OAX SSC
- Add 20ml 20X SSC to 950 ml purified water. Adjust pH to
7.0 with IN NaOH. Add purified water to bring final volume of the solution to 1 liter. Store up to six months at
room temperature. The washing solution must be at
74°C before using. In order to maintain the proper temperature, four slides must be placed in the heated solution
at one time. If fewer than four slides have been hybridized,
room temperature microscope slides must be used to bring
the number to four slides. If more than four slides have
been hybridized they must be washed in more than one
Coplin jar. The temperature of the wash solution must return to 74°C before washing each jar. Do not remove the
coverslips from several slides before placing any of the
slides in the wash jar. Begin timing the incubation when
the last slide has been added to the wash bath.
Fluorescent
staining
reagents
Recipes