Hoechst 33348

Seminal fluid was collected from the cauda epididymides of male mice at 8 months of age and the weight was measured. The sperm were incubated with the medium (FERTIUP Kyudo Co. Ltd. Saga, Japan) containing Hoechst 33348 (1.5μg/ml, Sigma Aldrich) for 1 hour. After incubation, the medium containing sperm was diluted with PBS and the number of sperm was counted. For analysis of sperm morphology, the sperm was observed by fluorescence microscope (All-in-one Fluorescence Microscope BX-X710, KEYENCE, Osaka, Japan). Further, the diluted medium containing sperm was smeared to slide glasses and the smears were stained with Wright-Giemsa and analyzed with a Zeiss Axioskop 2 plus microscope (Carl Zeiss, Tokyo, Japan) with a Olympus DP74 camera (Olympus Co., Tokyo, Japan).

Larvae were bisected in ice cold PBS. Fat bodies were isolated from carcasses, washed in PBS, and incubated for 30 min in a solution of Lysotracker DND-99 (Thermo Fisher, Waltham, MA, USA) 1:3000 in PBS and Hoechst 33348 (Sigma-Aldrich), fats were washed and then photographed under a Zeiss LSM510 confocal microscope (Zeiss, Germany).