15 Electron Microscopic in Situ Hybridization and its Combination
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and thereafter developed with 0.017% H20 2 in DAB solution
for 7 mins.
11. 2% osmium tetroxide is applied to the sections. After dehydration with graded ethanol (50%, 70%, 80%, 90%, 95%,
100%), tissue sections are embedded in Epon resin by inverted beam capsule method.
12. Polymerization at 60°C for 2 days. Ultrathin sections are inspected under an electron microscopy.
The negative control experiments: hybridization with probes of
sense or scramble sequence and without probes.
Postembedding method
1. Tissues are fixed at 4°C overnight in 4% paraformaldehyde
dissolved in PBS, and embedded in LR White resin. Tissues
are carefully placed at the bottom of 00 gelatin capsules,
which are filled with LR White resin and sealed.
2. After polymerization at 50°C for 24 hrs in a vacuum oven,
ultrathin sections are retrieved on Nickel grids.
3. After prehybridization at 37°C for 30 mins, hybridization is
carried out on the grids at 37°C overnight. The concentration
of the biotinylated oligonucleotide probe is 1 ng/Ill.
4. After hybridization, the grids are dipped in 2xSSe, lxSSC
and then O.5xSse for 5 mins each.
5. The hybridization signals are developed for 30 mins with 20
nm streptavidin gold diluted 1:50 in 1% BSA-PBS. After
being dipped in PBS and distilled water and dried at
room temperature, the grids are inspected under an electron
microscopy.
The negative control experiments: hybridization with probes of
sense or scramble sequence and without probes.