130
HIROTAKA MATSUMI AND TETSU YANO
- Formamide (Wako) (for posthybridization wash); Formamide used for posthybridization wash does not need to be
deionized.
- Denhardt's solution; [0.02% BSA, 0.02% Ficoll-400
(MW=400000), 0.02% polyvinylpyrolidone (MW=360000)]
- Yeast tRNA (Sigma)
- Salmon testis DNA (Sigma); Autoclave to fragments of 100 -
300 bp and store in aliquots at - 20°C.
- Dextran sulfate (MW=500000)
- 20x SSC (Ix ssc = 0.15 M NaCl, 0.015 M sodium citrate, pH
7.0)
- Bovine serum albumin (BSA, 98-99 % pure, Sigma)
- Normal goat IgG (Sigma)
- Horseradish peroxidase (HRP)-labeled mouse anti-T -T antibody (Kyowa Medex).
Notes • 4 % PFA/PBS; Dissolve 4 g ofPFA in 80 ml of Ix PBS with heat,
keeping the temperature between 55 - 60°C. Stir the slurry
until the powder is completely dissolved, and, if necessary,
add a few drops of ION NaOH to clear the solution. Adjust
the volume to 100 ml and store at 4 C before use. Four %
PF A/PBS is usually prepared freshly before use, but can be
stored at 4 °c for up to 2 weeks.
• The constituents of hybridization mixture; 10 mM Tris/HCl
buffer (pH 7.4), 0.6M NaCI, 1mM EDTA, 1 X Denhardt's solution, 250 ).lg/ml yeast tRNA, 125 ).lg/ml salmon testis DNA, 10
% dextran sulfate, 40 % deionized formamide and 0.5 - 4 ).lg/
ml T -T dimerized ds cDN A probe. It is denatured for 10 mins
before hybridization.
• The blocking solution contains 500).lg/ml normal mouse IgG,
5 % BSA, 0.3 M NaCI, 100).lg/ml salmon testis DNA, 100).lg/ml
yeast tRNA in PBS.
• The chromogen solution contains 3, 3-diaminobenzidine tetrahydrochloride (DAB), H20 2, nickel and cobalt; 0.5 mg/ml
DAB/0.025 % CoCh /0.02 % NiS04(NH4hS04/ 0.01 %
H20 2/O.1 M phosphate buffer (pH 7.5)
HIROTAKA MATSUMI AND TETSU YANO
- Formamide (Wako) (for posthybridization wash); Formamide used for posthybridization wash does not need to be
deionized.
- Denhardt's solution; [0.02% BSA, 0.02% Ficoll-400
(MW=400000), 0.02% polyvinylpyrolidone (MW=360000)]
- Yeast tRNA (Sigma)
- Salmon testis DNA (Sigma); Autoclave to fragments of 100 -
300 bp and store in aliquots at - 20°C.
- Dextran sulfate (MW=500000)
- 20x SSC (Ix ssc = 0.15 M NaCl, 0.015 M sodium citrate, pH
7.0)
- Bovine serum albumin (BSA, 98-99 % pure, Sigma)
- Normal goat IgG (Sigma)
- Horseradish peroxidase (HRP)-labeled mouse anti-T -T antibody (Kyowa Medex).
Notes • 4 % PFA/PBS; Dissolve 4 g ofPFA in 80 ml of Ix PBS with heat,
keeping the temperature between 55 - 60°C. Stir the slurry
until the powder is completely dissolved, and, if necessary,
add a few drops of ION NaOH to clear the solution. Adjust
the volume to 100 ml and store at 4 C before use. Four %
PF A/PBS is usually prepared freshly before use, but can be
stored at 4 °c for up to 2 weeks.
• The constituents of hybridization mixture; 10 mM Tris/HCl
buffer (pH 7.4), 0.6M NaCI, 1mM EDTA, 1 X Denhardt's solution, 250 ).lg/ml yeast tRNA, 125 ).lg/ml salmon testis DNA, 10
% dextran sulfate, 40 % deionized formamide and 0.5 - 4 ).lg/
ml T -T dimerized ds cDN A probe. It is denatured for 10 mins
before hybridization.
• The blocking solution contains 500).lg/ml normal mouse IgG,
5 % BSA, 0.3 M NaCI, 100).lg/ml salmon testis DNA, 100).lg/ml
yeast tRNA in PBS.
• The chromogen solution contains 3, 3-diaminobenzidine tetrahydrochloride (DAB), H20 2, nickel and cobalt; 0.5 mg/ml
DAB/0.025 % CoCh /0.02 % NiS04(NH4hS04/ 0.01 %
H20 2/O.1 M phosphate buffer (pH 7.5)