18 Electron Microscopic in Situ Hybridization and its Combination
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1. Fresh frozen specimens: Immediately after resection, specimens (size;5-10 mm) were embedded in OCT compound
and frozen under dry ice added ethanol. The frozen sections
were stored under -80°C.
2. Dry up for fresh frozen section: (5-6Ilm) fresh frozen sections were dried up on gelatin coated slide glass.
3. Fixation: 4% PFA (10 mins 4°C)
4. PBS (RT, 5 min.x3)
5. Incubation: 50 mM Tris/HCI(pH7.4)/5% non-fat dry milk/
50 mM NaCI/l mM EDTA (RT, 1 hr)
6. Incubation: T-T dimerized double strand DNA(1.0-2.5Ilg/
ml)/50 mM Tris/HCI(pH7.4)/5% non-fat dry milk/50 mM
NaClIl mM EDTA (RT, overnight)
7. Washing: 50 mM Tris/HCI(pH7.4)/5% non-fat dry milk/50
mM NaClIl mM EDTA (RT,15 mins x2) PBS (RT,15mins x3)
8. Blocking: 500 Ilg/ml goat IgG/5% BSA/100 Ilg/ml salmon testis DNA/lOO Ilg/ml yeast tRNA/NaN3/PBS (RT,1 hr)
9. First antibody: Rabbit anti-T-T IgG / 5% BSA/lOO Ilg/ml salmon testis DNA/lOO Ilg/ml yeast tRNA/NaN3/PBS (RT, overnight)
10. PBS (RT, 15 mins x4)
11. Second antibody: HRP-goat anti-rabbit IgG/5% BSA/lOO Ilg/
ml salmon testis DNA/lOO Ilg/ml yeast tRNA/NaN3/PBS (RT,
1 hr)
12. PBS (RT, 15 mins x4)
13. Visualization: 0.5 mg/ml DAB/0.025% CoCI2/0.02% NiS04(NH4)zS04 /0.01 % H20 2 /O.IM sodium phosphate buffer(pH7.2) (RT, 5 mins)
Procedure of Southwestern histochemistry (using digoxigenin
(Dig) labeled ds oligonucleotides):
1. Fresh frozen specimens: Immediately after resection, specimens (size;5-1O mm) were embedded in OCT compound
and frozen under dry ice added ethanol. The frozen sections
were stored under -80°C.