Biomedical Engineering Reference
In-Depth Information
9.7
Post-hybridization Washing Treatment
1. Prepare solutions and set each container (Fig.
9.5
).
2. To remove mineral oil, immerse the metal rack into at least two consecutive
chloroform washes. Between transfers of chloroform washes, dip the solution
off the rack very well.
3. Place the metal rack with slides into 2× SSPE + 0.1 %
-mercaptoethanol.
4. Remove the cover slips from the slides with forceps while the slides are in this
solution and quickly.
5. Place slides into a metal rack in 2× SSPE + 0.1 %
β
β
-mercaptoethanol without
letting the section dry {
Note *1
}.
6. Incubate slides in 2× SSPE + 0.1 %
β
-mercaptoethanol for 30 min at RT with
occasional shaking {
Note *1
}.
7. Incubate slides in pre-warmed 50 % formamide/2× SSPE/0.1 %
β
-mercaptoethanol for 1 h at 65 °C with occasional (two to three times) shaking
{
Note *1
}.
8. Incubate in pre-warmed 0.1× SSPE/0.1 %
β
-mercaptoethanol for 30 min at
65 °C with occasional shaking {
Note *1
}.
9. Repeat the same procedure (0.1× SSPE/0.1 %
β
-mercaptoethanol for 30 min at
65 °C) again in another new solution.
10. Run through the alcohol series, 50 % EtOH, 70 % EtOH, 95 % EtOH, and
100 % EtOH, for 2 min each {
Note *2
}.
11. Let the slides dry under the hood on a paper towel.
Note
*1: After rinsing, this 2× SSPE + 0.1 %
β
-mercaptoethanol must be handled as
radioactive waste.
*2: These alcohol solutions can be reused over 10 times, with ~60 slides per time.
Fig. 9.5
Solution series for post-hybridization washing
