9 In Situ Hybridization for RNA: Nonradioactive Probe: ds eDNA Probe
135
13. Wipe each slide around the tissue and react with 30-40 III of
the antibody (HRP-labeled mouse anti-T -T antibody) on
each slide for 12 h in a moist chamber humidified with PBS.
14. Wash 4 times with 0.075 % Brij 35/PBS for 15 mins each.
15. After a brief washing with PBS, visualize the HRP sites in a
chromogen solution containing DAB, H20 2, nickel and cobalt.
16. 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.
• The slide should be wiped with gause as well as possible not to
overdilute solutions. However, the slides must not be allowed
to dry out.
• 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 formam ide
is increased to a level as high as 80 %, and 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.
• The volume of hybridization mixture can be increased for
large sections.
135
13. Wipe each slide around the tissue and react with 30-40 III of
the antibody (HRP-labeled mouse anti-T -T antibody) on
each slide for 12 h in a moist chamber humidified with PBS.
14. Wash 4 times with 0.075 % Brij 35/PBS for 15 mins each.
15. After a brief washing with PBS, visualize the HRP sites in a
chromogen solution containing DAB, H20 2, nickel and cobalt.
16. 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.
• The slide should be wiped with gause as well as possible not to
overdilute solutions. However, the slides must not be allowed
to dry out.
• 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 formam ide
is increased to a level as high as 80 %, and 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.
• The volume of hybridization mixture can be increased for
large sections.