Fat bodies were carefully dissected from 5 mid-third instar larvae (72 hours after hatching, hAH) or white prepupae (96 hAH) in 4% PFA/PBS, briefly rinsed with 4% PFA/PBS, and pooled in 200 µ l of methanol (Fisher chemical, A452–4) on ice. The fat bodies were thoroughly homogenized using pestles with a micro-tube homogenizer. After centrifugation at 4°C for 10 min, the supernatant was pooled on ice, while the pellet was re-extracted by 100 µ l of methanol. The resulting extract was stored at −20°C until use. For hemolymph samples, mid-third instar larvae (72 hAH) or white prepupae (96 hAH) were rinsed in PBS, and dried on tissue paper. The cuticle was carefully torn to release the hemolymph onto a parafilm membrane. 2 µl of hemolymph were collected from 5 mid-third instar larvae or white prepupae and mixed with 200 µ l of methanol on ice. After vortexing, samples were centrifuged at 4°C for 10 min, and the resulting supernatant was stored at −20°C until use.
The sample solutions were dried with a CentriVap concentrator (Labconco) and dissolved in EIA buffer (Cayman Chemical). 20E AChE tracer, 20E EIA antiserum, Precoated (Mouse Anti-Rabbit IgG) ELISA 96-well strip plate, Wash buffer, and Ellman’s reagent were all purchased from Cayman Chemical. The assay was performed according to the manufacturer’s instructions using synthetic 20E (Sigma-Aldrich) as a standard.
The sample solutions were dried with a CentriVap concentrator (Labconco) and dissolved in EIA buffer (Cayman Chemical). 20E AChE tracer, 20E EIA antiserum, Precoated (Mouse Anti-Rabbit IgG) ELISA 96-well strip plate, Wash buffer, and Ellman’s reagent were all purchased from Cayman Chemical. The assay was performed according to the manufacturer’s instructions using synthetic 20E (Sigma-Aldrich) as a standard.