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YUKA IWASAKI AND IHACHI KA TO
- The constituents of prehybridization mixture; 10 mM Tris/
HCI buffer (pH 7.4), 0.6 M NaCl, 1 mM EDTA, 1 x Denhardt's
solution, 500 Ilg/ml yeast tRNA, 250 Ilg/ml salmon testis DNA,
40% deionized formamide a ) and Illg/ml digoxigenin-Iabeled
oligo-DNA probe. It is heat-denatured for 7 mins before hybridization.
a) Formamide is deionized with mixed bed resin (formamide
50 ml + mixed bed resin 2.5 g).
- The constituents of hybridization mixture; 10 mM Tris/ HCI
buffer (pH 7.4), 0.6 M NaCI, 1 mM EDTA, 1 x Denhardt's solution, 250 Ilg/ml yeast tRNA, 125 Ilg/ml salmon testis DNA,
10% dextran sulfate, 40% deionized formamide a ) and 0.5-2
Ilg/ml digoxigenin-Iabeled oligo-DNA probe. It is heat-denatured for 7 mins before hybridization.
a) Formamide is deionized with mixed bed resin.
- The blocking solution containing 500 Ilg/ml normal sheep
IgG, 5% BSA, 100 Ilg/ml tRNA, 100 Ilg/ml salmon testis
DNA in PBS
- The chromogen solution containing 3,3' -diaminobenzidine
(DAB), HzOz, nickel and cobalt; 0.5 mg/ml DAB/0.025%
CoClz/0.02% NiS04 (NH4hS04/0.01% HzOz/O.l M phosphate
buffer (pH 7.5)
for In situ - PFA
hybridization - proteinase K (Wako)
- formamide (nacalai tesque)
- mixed bed resin [AG-501-X8 (D) ; Bio-Rad]
- Denhardt's solution [0.02% BSA (98-99% pure), 0.02% Ficoll400 (MW=400000), 0.02% polyvinylpyrolidone (MW =
360000)]
- yeast tRNA
- salmon testis DNA
- dextran sulfate (MW=500000)
- 20 x SSC
- normal sheep IgG
- HRP-Iabeled sheep anti-digoxigenin antibody
- The constituents of hybridization mixture; 10 mM Tris/ HCI
buffer (pH 7.4),0.6 M NaCl, 1 mM EDTA, 1 x Denhardt's solution, 250 Ilg/ml yeast tRNA, 125 Ilg/ml salmon testis DNA,
10% dextran sulfate, 40% deionized formam ide and 0.5-2Ilg/
ml digoxigenin-labeled oligo-DNA probe. It is heat-dena-