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.