12 In Situ Hybridization for RNA: Nonradioactive Probe: Oligo-DNA Probe
173
- oligo-DNA probe
for Oligo-DNA
- digoxigenin (DIG) oligonucleotide tailing kit (Boehringer tailing
Mannheim)
- UV lamp (254 nm wave length) (Toshiba Ltd., Tokyo, Japan)
- UV radiometer (taking an accurate measurement of 5 J/mzl
sec)(Tokyo, Kougaku Kikai, Ltd., Tokyo, Japan)
- nitrocellulose filter
- vinyl bag
- 20 x SSC (1 x ssc = 0.15 M NaCl, 0.015 M sodium citrate, pH
7.0)
- bovine serum albumin (BSA, 98-99% pure) (Sigma)
- normal sheep IgG (Sigma)
- yeast tRNA (Sigma)
- salmon testis DNA (Sigma)
- horseradish peroxidase (HRP)-labeled sheep anti-digoxigenin antibody (Boehringer Mannheim)
- The blocking solution containing 500 /lg/ml normal sheep
IgG, 5% BSA, 100 /lg/ml yeast tRNA, 100 /lg/ml salmon testis
DNA in PBS.
- The chromogen solution containing 3,3' -diaminobenzidine
(DAB), HzOz, nickel and cobalt; 0.5 mg/ml DAB/O.025%
CoClz/0.02% NiS04 (NH4)zS04/0.01 % HzOz 10.1 M phosphate
buffer (pH 7.5). (Adams 1981)
for Immunohistochemical
detection of
probes on nitrocellulose
membranes
- UV lamp (254 nm wave length)
for Dot blot
- UV radiometer (taking an accurate measurement of 5 J/mz/ hybridization
sec)
- nitrocellulose filter
- vinyl bag
- 20x SSC
- 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) (Pharmacia Fine Chemicals), 0.02% polyvinylpyrolidone (MW=360000) (Sigma)]
- dextran sulfate (MW=500000) (Sigma)
- yeast tRNA
- salmon testis DNA
- BSA
- HRP-Iabeled sheep anti-digoxigenin antibody