20e acetylcholinesterase

Ecdysteroid titres were quantified by ELISA essentially62 (link). 20E (Sigma) and 20E-acetylcholinesterase (Cayman Chemicals) were used as the standard and enzymatic tracers, respectively. Absorbance was read at 415 nm using a microplate reader Model 680 (Bio-Rad). The ecdysteroid antiserum has the same affinity for E and 20E (ref. 63 (link)), but because the standard curve was obtained with the latter compound, the results are expressed as 20E equivalents. For sample preparation, 10–30 staged larvae were weighed and homogenized in 100 μl of methanol three times. After centrifugation at 14,000 r.p.m. for 5 min, supernatants were transferred to new tubes and dried with centrifugal evaporator. Samples were resuspended in 50 μl of EIA buffer (0.1M phosphate solution containing 0.1% BSA, 0.4 M NaCl, 1 mM EDTA and 0.01% NaN₃) and incubated at 4 °C overnight.

Flies were reared at 25°C and aged for 4 days. Ovaries were dissected in PBS. Samples were homogenized in 50 μL of methanol in 1.5-mL tubes using a pestle. After centrifugation at 20,913 x g for 1 min, the supernatants were transferred to new tubes and dried with a centrifugal evaporator. Samples were resuspended in 50 μL of EIA buffer (Cayman Chemicals) according to the manufacturer’s protocol and incubated at 4°C overnight. Ecdysteroid levels were quantified by an enzyme-linked immunosorbent assay using anti-20E antiserum (Cayman Chemicals) and 20E-acetylcholinesterase (Cayman Chemicals) as essentially described [62 (link)]. Note that the antiserum used in this study is known to recognize not only 20-hydroxyecdysone (20E) but also ecdysone [63 (link)]. In this study, we used we used 20E (ENZO Life Sciences) as a standard, and the ecdysteroid amount was expressed in 20E equivalents. Absorbance was measured at 415 nm using a microplate reader Multiskan GO (Thermo Fisher Scientific).