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Adult wild type, 1151>lbRNAi and 1151>lbe males were fixed on microscope slides in the "legs-up" position using a sealing solution prepared from double-sided 3M Scotch tape and N-heptane as a solvent.
In our mate choice tests, focal and audience males were separated as the audience males were fixed in a Plexiglas cylinder.
Elements of c associated with older males were fixed to 0. OCS coped because young males tended to have higher breeding values than older males.
Mating plugs dissected from recently mated A. gambiae females, and MAG dissected from A. gambiae, A. arabiensis, and A. merus virgin 4- to 5-day-old males were fixed in PBS 4% formaldehyde solution.
Six heads from 2- and 14-day-old ELAV-Gal4, UAS-GFP/UAS-X, and mutant males were fixed overnight at 4°C in 2% PFA plus 1% glutaraldehyde, postfixed in 1% osmium tetroxide (OsO4) at RT, dehydrated in an ethanol series, and embedded in Epon 812.
Elements of c associated with all other males were fixed to 0. In the breeding scheme sire multiples, elements of c associated with male candidates were restricted to 0, x/600, 2 x/600, … 0.5, where x is the sire multiple (x = 2, 5, 10, 20, 50, and 100).
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However, the palps of dead males are fixed in an inflated state, making them hard to remove.
The proportion of wild-type and GFP males was fixed in this F0 generation (m), following the expected average proportion of males among EEViA1-6 observeduringng experimental evolution; reported in [ 29] (Additional file 1: Figure S1): for G0, G30, G60 and G100, male proportions were of 0, 0.15, 0.1, and 0.15, respectively.
For in situ hybridization, male antennae were fixed overnight at 4°C in 4% PFA-3% Tween 20, rinsed in PBS (0.85% NaCl, 1.4 mM KH2PO4, 8 mM Na2HPO4, pH 7.1; Sigma) and then cryoprotected in PBS-18% sucrose at 4°C before use.
After plants were scored for phenotypes, pWSRi SpPI male inflorescences were fixed, imbedded and thin sectioned.
Male testes were fixed in a 3 1 ethanol: acetic acid solution, and female gastric caeca were removed and fixed as described by Castillo et al. [ 63].
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