15 Electron Microscopic in Situ Hybridization and its Combination
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Materials
- Electron microscopy (e.g., JEOL 1200EX)
Equipment
- incubator
- vacuum oven
Ribonuclease-free solutions treated with 0.02% diethylpyrocar- Reagents
bonate (DEPC), autoclaved glass ware and gloves should be used
for in situ hybridization study.
- synthesized oligonucleotide probes
ENZO's terminal labeling kit (ENZO Diagnostics Inc., Farmingdale, NY, USA)
4% paraformaldehyde
0.01 M phosphate buffered saline pH 7.4 (PBS)
graded concentrations of sucrose dissolved in PBS (10%, 15%,
20%)
Optimal Cutting Temperature (OCT) compound (Tissue-Tek;
Miles Laboratories Inc., Elkhart, Ind., USA)
3-aminopropylmethoxysilane-coated slides
proteinase K
acetic anhydride
triethanolamine
2x sodium chloride sodium citrate (SSC)
prehybridization solution: 10% dextran sulfate, 3xSSC, Ix
Denhardt's solution (0.02% Ficoll/ 0.02% bovine serum albumin (BSA)/ 0.02% polyvinylpyrrolidone), 100 Ilg/ml salmon
sperm DNA, 125 Ilg/ml yeast tRNA, 10 Ilg/ml polyadenyliccytidylic acid, I mg/ml sodium pyrophosphate pH 7.4 and
50% formam ide
- Vectastain's ABC kit (Vector Laboratories Inc., Burlingame,
Ca., USA)
- diaminobenzidine (DAB) solution: 20 mg of freshly prepared
3,3' -diaminobenzidine tetrahydrochloride dissolved in 0.05
M Tris-HCl pH 7.6
- H20 2
- osmium tetroxide
- Epon resin (e.g., Quetol-812)
- LR White resin (Polyscience ; Warrington, P A)
- beam capsule
- 00 gelatin capsules (Lilly Pharmaceuticals; Indianapolis, IN)