EC-tagging and EU-tagging for RNA imaging
Larvae were fed 1 mM EC or 0.5 mM EU for the indicated times. For
EU-tagging in the presence of RNA polymerase inhibitors, dissected
brains were incubated in D22 media containing 0.5 mM EU for four hours.
Drug treated brains were pre-incubated in the presence of the inhibitor
for two hours prior to addition of EU, control brains were pre-incubated
in media alone. Triptolide (ThermoFisher) was used at a final
concentration of 100 µM, 10-fold higher than the concentration known to
inhibit RNA polymerase II in Drosophila tissue culture cells
[25]. We confirmed this concentration blocks production of mRNA in
cultured brains using a dot blot (data not shown). Actinomycin D
(Millipore Sigma) was used at a final concentration of 700 µM, a
concentration that is expected to affect RNA polymerase I and II
[21]. For all imaging experiments, brains were fixed in 4%
paraformaldehyde prior to Alexa Fluor 488 addition using the Click-iT
RNA Imaging Kit (ThermoFisher) as previously described for Click-iT
kit-based detection of DNA labeled with 5-ethynyl-2’-deoxyuridine inDrosophila larval brains [37]. The Click-iT reaction was
followed by antibody staining according to standard methods [38].
Imaging was performed using a Zeiss LSM 880 confocal microscope.