The flap was immediately thereafter washed in sterile H2O2 and NaCl 9 mg/ml dried with sterile gauze, packed in a two-layer sterile instrument pouch (3 M Health Care 9097 St . Paul MN 55144 1000 USA), and placed in an ultra-low freezer (Forma Scientific Inc. SA mod. 925, 401 Mill Creek Rd, Marietta, OH 45750) at - 86 °C.
PBMCs were immediately fixed thereafter using fixation/permeabilization buffer (True-Nuclear-kit, BioLegend) according to manufacturer's instructions.
Cells treated with 25 ng/mL Emmprin or 100 µM deferoxamine (DFO) for 4 hours were immediately fixed; thereafter subjected to in situ PLA using Duolink Detection kit (Olink Bioscience, Uppsala, Sweden) according to the manufacturer's instructions for Duolink Blocking solution and Detection protocol.
Thereafter, samples were immediately centrifuged and treated as above.
Samples were immediately stored at −20 °C and thereafter simultaneously analyzed by pyrosequencing.
Thereafter, tissue cultures were immediately fixed.
The pressure was immediately released thereafter.
Thereafter, they were surface dried and the weights were immediately measured.
Thereafter quercetin was mixed into the meal of four animals and meals were immediately fed.
Alternatively, the cells were post-incubated without Z-DEVD but were exposed immediately thereafter to annexin V (BioVision Research Products, Mountain View, CA, USA) at the concentration of 0.2 mg ml−1 in annexin V binding buffer (PharMingen BD Biosciences, Mississauga, Ontario, Canada) for 30 min at room temperature.
F1 males were collected, housed three days post-eclosion, and nucleic acids were extracted immediately thereafter from either testes or whole fly body.
