Biomedical Engineering Reference
In-Depth Information
*4: Preparation of in situ hybridization solution.
For total 10 mL volume:
Mix 5 mL of 100 % formamide + 600
μ
L of 5 M NaCl + 100
μ
L of 1 M Tris-
HCl pH 8.0 + 240
μ
L of 0.5 M EDTA pH 8.0 + 200
μ
L of 50× Denhart's solu-
tion + 100
L of 20 mg/mL tRNA [Roche,
cat#109495] + 1 g of sodium dextran sulfate 500,000.
Finally, bring volume up to 10 mL with pure water.
Much time is needed to dissolve sodium dextran sulfate by shaking at
room temperature.
Stock the solution in −20 °C.
μ
L of 1 M DTT + 250
μ
9.6
Pre-hybridization and Hybridization
1. Prepare appropriate amounts of 4 % paraformaldehyde/1× PBS {
Note*1
}, 1×
PBS {
Note*2
}, and 2× SSPE {
Note*3
}, and set each container (Fig.
9.3
).
2. Immerse glass slides with tissue sections in 4 % paraformaldehyde/1× PBS for
5 min at room temperature (RT) {
Note*4
}.
3. Rinse three times in 1× PBS in three separate containers, 2 min each, with occa-
sionally gentle shaking.
4. Put slides in 1 L of acetylation solution {
Note*5
} for 10 min.
5. Rinse three times in 2× SSPE for each 2 min in three separate containers.
6. Dehydrate through the alcohol series, 50 % EtOH, 70 % EtOH, 95 % EtOH,
and then 100 % EtOH {
Note*6
}, for 2 min each.
7. Let the slides dry under the hood on a paper towel.
8. Calculate the total volume needed for all slides (plus a few extra), from 50 to
150
l {
Note*7
} of hybridization solution per slide.
9. Mix S
35
-cRNA probe and hybridization solution very well, but gently, avoiding
generation of air bubbles (adjust 10
6
cpm S
35
-cRNA probe/100
μ
μ
L hybridiza-
tion solution).
Fig. 9.3
Solution series for pre-hybridization
