Labeling and hybridization
DNA probes were generated from 5 μg total RNA by reverse transcriptase synthesis (Superscript II enzyme, Life Technologies), using oligo-dT primers and biotin-labeled dUTP and dATP (in a 1:10 molar ratio to unmodified dTTP and DATP, respectively). Probes were cleaned by spin filtration, denatured, and then incubated in hybridization buffer (50% formamide, 6 × SSPE, 0.5% SDS, 5 × Denhardt's solution) with 0.1 pmol poly-(A)25 for 2 h at 42°C. Hybridization was carried out at 42°C, followed by a series of stringency washes, as in [3]. Membranes were washed, bathed in the Avidx alkaline-phosphatase conjugate then exposed to the chemiluminescent substrate CDP-star, according to the manufacturer's instructions (Tropix, Applied Biosystems). Membranes were next exposed to autoradiographic film for between 10 min and 8 h. Each membrane was used only once, giving an independent hybridization for 2 embryo replicates, 4 bipotential larvae, 13 queen, and 13 worker larval samples.