Vectashield fluorescent medium

Larval offspring resulting from crosses between the heterozygous AcIr76b-QF2 driver line and the QUAS-mCD8:GFP effector were screened for eye-specific expression of DsRed. Larval head appendages as well as whole adult antennae, maxillary palps, proboscis, and tarsi from 4- to 6-d-old adult females were dissected into 4% formaldehyde in PBST (0.1% Triton X-100 in phosphate-buffered saline) and fixed on ice for 30 min in the case of the tarsi and larval head appendages or at 4 °C for 24 h for adult antennae. Samples were thereafter washed 3X in PBST for 10 min each and directly transferred onto Superfrost plus slides (VWR Scientific) and mounted in Vectashield fluorescent medium (Vector Laboratories). Confocal microscopy images at 1024 × 1024 pixel resolution were collected with an Olympus FV-1000 instrument equipped with a 100× oil objective at the Vanderbilt University Cell Imaging Shared Resource Core. Laser wavelengths of 405 nm, 488 nm, and 543 nm were used to detect DAPI, green fluorescent protein (GFP), and Cy3, respectively.

MDA-MB-231 human breast cancer cells were cultured in minimum essential medium (Hyclone; Thermo Fisher Scientific Inc) containing 10 μM H₂-DCFDA in a humidified incubator at 37°C for 30 minutes. Cells were washed in PBS (pH 7.4) and lysed in lysis buffer (25 mM HEPES [pH 7.4], 100 mM NaCl, 1 mM EDTA, 5 mM MgCl₂, and 0.1 mM dithiothreitol [DTT], supplemented with a protease inhibitor cocktail). Cells were cultured on coverslips in a four-well plate. Cells were incubated in DMEM containing 10 μM H₂-DCFDA at 37°C for 30 minutes. Cells were washed in PBS, mounted with Vectashield fluorescent medium (Vector Laboratories, Burlingame, CA, USA), and viewed with a fluorescence microscope.