Biomedical Engineering Reference
In-Depth Information
Fig. 9.4
Cover slip procedure and hybridization in oil bath with radioactive RNA probe. (
a
) Setting
of cover slip procedure. (
b
) Labeling sample information on glasses with a pencil. (
c
) Putting S35-
cRNA probe/hybridization solution on a cover slip. (
d
&
e
) Placing a glass slide on a cover slip, then
immediately fl ip the side to face up. (
f
&
g
) Placing the cover-slipped slides in a metal rack. (
h
)
Placing the metal racks into the oil bath
10. Incubate mixed solution at 65 °C water bath for 5 min to dissolve reagents, and
then immediately chill on ice at least for 5 min.
11. Put S
35
-cRNA probe/hybridization solution on a cover slip, and gently place a
glass slide on it with the tissue sections facing the hybridization solution
(Fig.
9.4a-e
).
12. Place the cover-slipped slide in a metal rack, making sure the slides stay in a
horizontal position (Fig.
9.4f-g
).
13. Carefully place each rack into the oil bath {
Note*8
} at 65 °C {
Note*9
}
(Fig.
9.4h
).
14. Incubate slides overnight (12-15 h) {
Note*10
}.
Note
*1: Preparation of 3 % paraformaldehyde/1× PBS:
For 1 L solution:
Mix 30 g paraformaldehyde in 100 mL of 10× PBS + 900 mL deionized
H
2
O (dH
2
O, no need to use DEPC-H
2
O), and add 320
μ
L of 10 N NaOH (to
help paraformaldehyde to dissolve, adjustment of pH).
While stirring, heat the solution (~40 °C) under a hood on the hot plate.
*2: Preparation of 1× PBS:
ten times dilution from 10× PBS.
