Hoechst blue

Salivary glands were dissected from non-viruliferous four-instar planthopper nymphs in cold distilled water on a glass plate, and fixed in 4% paraformaldehyde for 2 hr at room temperature. After being permeabilized with osmotic buffer (0.01 M phosphate-buffered saline containing 2% Triton X-100, pH 7.4) for 4 hr, the salivary glands were blocked with 1% bovine serum albumin for 30 min at room temperature. The samples were incubated with the primary antibody, anti-human GPS2 goat polyclonal antibody (Santa Cruz Biotechnology), overnight at 4°C. After washing with 0.01 M phosphate-buffered saline containing 1% Tween-20 (pH 7.4), the secondary antibody, Alexa Fluor 594 (red) affinipure donkey anti-goat IgG (YEASEN, Shanghai, China), was added. The nuclei were counterstained with Hoechst (blue) in accordance with the manufacturer’s instructions (Invitrogen). Negative control was without the primary antibody. The images were viewed under a Leica TCS SP5 confocal microscope (Leica Microsystems, Solms, Germany). Twenty salivary glands were tested.

AML12 cells were fixed with 4% paraformaldehyde (diluted from 16%, Alfa Aesar, 43368-9M) and stained with Hoechst blue (Invitrogen, 953557) and Nile red (Invitrogen, N1142). Two images (Zeiss Axiovert 200 with QICAM Fast 1394), one of 4′,6-diamidino-2-phenylindole (DAPI) and one of Nile red, were taken at three locations in each well. Lipid droplet size and quantity were calculated from epifluorescent widefield micrographs using an ImageJ plugin, MRI Lipid Droplets (75 ). Lipid droplets were identified as areas larger than five pixels. MRI Lipid Droplets ImageJ plugin was used to identify nuclei in DAPI staining and count total cell number.