9 In Situ Hybridization for RNA: Nonradioactive Probe: ds cDNA Probe
137
13. Wipe each slide around the tissue and react with 30-40 III of
the 1st antibody (anti-T -T mouse IgG) for 12 hrs in a moist
chamber humidified with PBS.
14. Wash 4 times with 0.075 % Brij 35/PBS for 15 mins each.
15. Wipe each slide around the tissue and react with 30-40 III of
the 2nd antibody (HRP -labeled goat anti -mouse IgG) for 1 hr
in a moist chamber humidified with PBS.
16. Wash 4 times with 0.075 % Brij 35/PBS for 15 mins each.
17. After a brief washing with PBS, visualize the HRP sites in a
chromogen solution containing DAB, H20 2, nickel and cobalt.
18. Dehydrate with serial up-graded ethanol solutions, cleared
with xylene and mounted without any counterstaining.
• Proteinase K solution is incubated in a water bath at 37°C for Notes
30 mins before use. Because proteinase K tends to change in
the activity, it is stored at -20°C in small aliquots, which are
used once and discarded. The effect of proteinase K digestion
varies with the tissue and type of fixation, so the concentration of proteinase K should be optimized with an individual
experiment.
• Salmon sperm DNA and yeast tRNA are added to inhibit
DNase and RNase, respectively. Furthermore, the addition
of 200U/ml heparin and/or 0.01 % SDS to the hybridization
mixture often decreased the nonspecific reaction of the
probes.
• If necessary, in order to obtain more stringent condition for
washing after hybridization, the concentration of formamide
is increased to a level as high as 80 %, the salt concentration is
decreased to a level as low as that of O.lx Sse. It is better to
perform hybridization at less than 50°C to preserve the morphology of tissues.
137
13. Wipe each slide around the tissue and react with 30-40 III of
the 1st antibody (anti-T -T mouse IgG) for 12 hrs in a moist
chamber humidified with PBS.
14. Wash 4 times with 0.075 % Brij 35/PBS for 15 mins each.
15. Wipe each slide around the tissue and react with 30-40 III of
the 2nd antibody (HRP -labeled goat anti -mouse IgG) for 1 hr
in a moist chamber humidified with PBS.
16. Wash 4 times with 0.075 % Brij 35/PBS for 15 mins each.
17. After a brief washing with PBS, visualize the HRP sites in a
chromogen solution containing DAB, H20 2, nickel and cobalt.
18. Dehydrate with serial up-graded ethanol solutions, cleared
with xylene and mounted without any counterstaining.
• Proteinase K solution is incubated in a water bath at 37°C for Notes
30 mins before use. Because proteinase K tends to change in
the activity, it is stored at -20°C in small aliquots, which are
used once and discarded. The effect of proteinase K digestion
varies with the tissue and type of fixation, so the concentration of proteinase K should be optimized with an individual
experiment.
• Salmon sperm DNA and yeast tRNA are added to inhibit
DNase and RNase, respectively. Furthermore, the addition
of 200U/ml heparin and/or 0.01 % SDS to the hybridization
mixture often decreased the nonspecific reaction of the
probes.
• If necessary, in order to obtain more stringent condition for
washing after hybridization, the concentration of formamide
is increased to a level as high as 80 %, the salt concentration is
decreased to a level as low as that of O.lx Sse. It is better to
perform hybridization at less than 50°C to preserve the morphology of tissues.