The following protocol was generously provided by Prof. Mindy George-Weinstein, Department of Anatomy, Philadelphia College of Osteopathic Medicine, 4170 City Avenue, Philadelphia, PA 19131
| Step | Action |
Timing
|
| 1 | Treat paraffin-embedded samples in Xylene #1 Fresh |
10
min
|
| 2 | Transfer to Xylene #2 Fresh |
10 min
|
| 3 | Transfer to 100% EtOH Fresh |
2
min
|
| 4 | Hydrate in 100% EtOH |
2
min
|
| 5 | Hydrate in EtOH: 90% - 80% - 70% - 50% - 30% |
5
min each
|
| 6 | Rinse in 0.85% NaCl |
10
min
|
| 7 | Rinse is 1X PBS + DEPC |
1
min
|
| 8 | Fix with 4% paraformaldehyde + DEPC |
20
min
|
| 9 | Dehydrate in EtOH: 70% - 90% - 100% |
5
min each
|
| Step | Action |
Timing
|
| 1 | Treat with 0.1%Triton+DEPC |
10
min
|
| 2 | Rinse with 1X PBS+DEPC |
2
min
|
| 3 |
Incubate with 0.1% Pepsin in 0.01M HCl @ room temp. |
10
min
|
| 4 | Rinse with 1X PBS+DEPC |
3
min
|
| 5 | Postfix with 1% Paraformaldehyde+DEPC |
10
min
|
| 6 | Dehydrate in EtOH. 70% - 90% - 100% |
5
min each
|
| 7 | Air dry slides |
| Step | Action |
Timing
|
| 1 | Put needed
amount of dendrimers into small tubes - Place in boiling water to denature. - Centrifuge at 4oC |
2 min 5 min |
| 2 | Place yeast/target RNA into 80oC oven |
10
min
|
| 3 | To 1500ul
of fresh Hybridization buffer add: - 75ul of SSS DNA - 75 ul yeast RNA |
|
| 4 | Dilute each 3DNA Dendrimer probe to be used in Hybridization buffer/RNA/DNA to a final concentration of 300 pg/ul | |
| 5 | Add 30ul of Dendrimer+Hybrid. Buffer to respective wells. | |
| 6 | Cover with autoclaved siliconized coverslips | |
| 7 | Place slides on 20X SSC soaked filter paper in 100mm petri dish | |
| 8 | Place dish in 80oC oven with lid propped open on one side |
10
min
|
| 9 | Place dish in 37oC incubator, let sit overnight |
18.5
hours
|
| 10 | Place
slides in 100mm petri dish with 15mls 1X PBS+DEPC Let coverslips float off and then remove |
15 min |
| 11 | Incubate slides in 60% Formamide in 2X SSC buffer |
10
min
|
| 12 | Counterstain with Biz-Benzamide diluted 1:1000 DH2O | |
| 13 | Put 1 drop of Gelmount medium on each slide well, coverslip with large single coverslip. Let sit for about 1 hour. |
1 hour |
| 14 | With filter paper, wick away excess mounting medium. Seal edges of Coverslip with nail polish. Store in dark |
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