9 In Situ Hybridization for RNA: Nonradioactive Probe: ds cDNA Probe
131
- Paraformaldehyde (PFA) (MERCK)
- Proteinase K (Wako); Dissolve the proteinase K at 1 mg/ml
concentration in DDW. Store at - 20°C in small aliquots.
- Mixed bed resin (Bio-Rad)
- Formamide (nacalai tesque) (for prehybridization); Formamide used for prehybridization must be of the highest grade
commercially available (nacalai tesque) and be deionized
with mixed bed resin just before use. (Mix 50 ml of formamide
and 5 g of mixed bed resin and stir for 30 mins.)
- 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) 1
- 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)
- Anti-T-T mouse IgG (Kyowa Medex)
- Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG
(Chemicon)
• 4 % PFA/PBS; Dissolve 4 gofPFA 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. 4 %
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/HCI
buffer (pH 7.4), 0.6M NaCl, 1mM EDTA, Ix 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 cDNA probe. It is denatured for 10 mins
before hybridization.
for in situ
hybridization
(indirect
method)
Notes
131
- Paraformaldehyde (PFA) (MERCK)
- Proteinase K (Wako); Dissolve the proteinase K at 1 mg/ml
concentration in DDW. Store at - 20°C in small aliquots.
- Mixed bed resin (Bio-Rad)
- Formamide (nacalai tesque) (for prehybridization); Formamide used for prehybridization must be of the highest grade
commercially available (nacalai tesque) and be deionized
with mixed bed resin just before use. (Mix 50 ml of formamide
and 5 g of mixed bed resin and stir for 30 mins.)
- 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) 1
- 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)
- Anti-T-T mouse IgG (Kyowa Medex)
- Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG
(Chemicon)
• 4 % PFA/PBS; Dissolve 4 gofPFA 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. 4 %
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/HCI
buffer (pH 7.4), 0.6M NaCl, 1mM EDTA, Ix 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 cDNA probe. It is denatured for 10 mins
before hybridization.
for in situ
hybridization
(indirect
method)
Notes