Whole larvae, bled carcasses or microdissected Malpighian tubules were collected in 100 µl of water, homogenized with a micropestle and sonicated.
Cells were then homogenized with a micropestle and centrifuged for 15 min at 4°C.
The radicle was thoroughly homogenized with a micropestle followed by incubation at 65°C for 30 min.
Before extraction, frozen samples were disrupted with a micropestle on dry ice followed by homogenization with the QIAshredder (Qiagen).
Freshly eclosed flies were resuspended in 50 μl extraction buffer (50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1 mM PMSF, 5 μg/ml leupeptin, 5 μg/ml E-64 and 1% Triton X-100) per fly and then thoroughly ground with a micropestle.
Six female flies were homogenized in 1000 μl TSB with a micropestle device (VWR, Radnor, PA) until pieces of tissue were no longer visible.
For the first two extraction protocols, the body part used was first dissected out, placed in a 1.5 mL microcentrifuge tube, frozen in liquid nitrogen and pulverized with a micropestle.
The tube was incubated at 65°C for 1 h and then kept on ice for 5 min. For filamentous fungi, the pellet was ground with a micropestle at the start and during the 65°C incubation.
For extract preparation, 0.5 mg tissue from tepals, anthers and stigma (collected at three different stages) was crushed with a micropestle in 700 μl Tris HCl (50 mM, pH 7.5 containing 1 M NaCl), and incubated for 10 minutes on ice.
John Mills, another veteran of starchy, upper-class parts, is a twinkling old buffer with a nose for "naughty salt".
