Slides were scanned using an Axon 4000B scanner, and images analyzed with GENEPIX software (Agilent Technologies, Santa Clara, CA). Spot finding by software was manually checked for all microarrays
Slides were passed quickly through steam and placed in a UV linker at 6000 x 100µJ/cm2. Before pre-hybridization, slides were plunged in 0.2% SDS and immediately shaken vigorously for 2 min. They were then washed twice in distilled water, transferred to 95% ethanol for 15 sec, and dried at 2000 rpm for 3 min. For hybridization, slides were incubated at 42o C in a Coplin jar for ~1h30, then washed in distilled water twice and isopraponol and dried at 2000 rpm for 3 min. Samples were incubated at 95-100o C for 3 min and then kept at 55oC until applied to the microarray slides. 50 ul of sample was applied on the slides and slides incubated for 18 h at 42∞C using a Maui Hybridization System (Kreatech).
Amplified RNA (2 µg) was labelled with a ULS aRNA labelling kit (Kreatech) according to kit directions. 60 ng labelled material from each sample was dried down and resuspended in 50 µL of hybridization solution.
Individual mushroom bodies were isolated as previously described (Sen Sarma et al., 2009). Frozen brains were treated overnight with RNAlater-ice (Ambion) and then dissected over ice. Only the calyces of the mushroom bodies were retained.
RNA extraction was carried out as indicated in the Qiagen RNeasy kit for total RNA with on-column DNase I treatment (Qiagen, Valencia, CA). To obtain sufficient RNA for single brain analysis, RNA (500ng) was amplified with the Amino Allyl MessageAmpô II aRNA Amplifcation kit (Ambion, Austin, TX), according to kit instructions (as in Whitfield et al. 2003, 2006). in vitro transcription proceeded with an incubation time of 6 h at 37∞ C.
1-day-old honey bee individuals were collected within 24 hours after emergence from the pupal stage. Some were frozen immediately for analysis, while the majority were marked and introduced to a natural honey bee colony. Bees were permitted to commence foraging behavior, and subsets of foraging honey bees were collected either 4, 8, 12 or 16 days after initiation of foraging behavior. Foraging honey bees were captured while returning to the colony entrance with a pollen load.