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AKIRA MATSUNO ET AL.
- sodium periodate
- 0.1 M phosphate buffer (PB)
- 20 nm protein A colloidal gold (EY Laboratories Inc., San
Mateo, Ca., USA)
- 20 nm streptavidin colloidal gold (Bio Cell Research Laboratories, Cardiff, UK)
Procedure
Preembedding method
1. Tissues are fixed at 4°C overnight in 4% paraformaldehyde
dissolved in PBS.
2. Tissues are immersed in graded concentrations of sucrose
dissolved in PBS at 4°C (10% for 1 hr, 15% for 2 hrs, 20%
for 4 hrs), and are embedded in OCT compound.
3. Six)lm thick tissue sections are mounted on 3-aminopropylmethoxysilane-coated slides.
4. After air drying for 1 hr, tissue sections are washed with PBS
for 15 mins.
5. Tissue sections are treated with 0.1 )lg/ml proteinase K in
PBS at 37°C for 30 mins, followed by treatment for 10
mins with 0.25% acetic anhydride in 0.1 M triethanolamine.
6. The slides are washed in 2x SSC at room temperature for 3
mins and then prehybridized with prehybridization solution
at 37°C for 30 mins.
7. The biotinylated probe with the concentration of 0.1 ng/)ll is
diluted with this prehybridization solution and hybridization is carried out at 37°C overnight.
8. The slides are washed at room temperature with 2xSSC,
1xSSC and then O.5xSSC for 15 mins each.
9. After being washed in PBS for 5 mins, the slides are dipped in
DAB solution for 1 hr.
10. The hybridization signals are detected with streptavidinbiotin-horseradish peroxidase, using Vectastain's ABC kit,