C2 si plus confocal microscope

To check the phagocytotic ability of hemocytes, we followed a previously described phagocytosis assay method [34 (link)]. Instead of using HmlΔ-Gal4 fly lines, we used HLT-Gal4 fly lines for the constant Gal4 expression in hemocytes. Escherichia coli BioParticle (Invitrogen P35361) and Staphylococcus aureus BioParticle (Invitrogen A10010) were used in separate assays. Larvae from 120 h AEL were bled and incubated in Schneider’s medium containing 1ug/ml BioParticle for 30 min at room temperature. Then, hemocytes were fixed with 3.7% formaldehyde for 30 min at room temperature on glass slides (Immuno-Cell Int.; 61.100.17) and washed 3 times in 0.4% Triton X-100 in 1x PBS for 10 min. After washing, samples were mounted in Vectashield and imaged using a Nikon C2 Si-plus confocal microscope. BioParticle uptake by hemocytes was counted using the IMARIS software (Bitplane).

Third instar lz-GAL4 >GFP; BcF6-mCherry larvae were heat killed in glycerol on a glass slide and directly mounted onto coverslip-bottom imaging dishes (ibidi, cat# 81158). The posterior hemocyte hubs were imaged with Z stacks of 2 μm distance each encompassing the entire area of the hub in a lateral direction using a Zeiss LSM 710 confocal microscope. Finally, all the stacks per larva were merged by summing the intensities on Fiji software for subsequent intensity measurements of GFP and mCherry.
Wasp infested w;UAS-GFPN-lacZ;btl-GAL4 larvae were imaged using Nikon C2 Si-plus confocal microscope.

HeLa cells were cultured in six-well plates with cover slips in each well (1.5 × 10⁴ cells/well). After cells were incubated overnight in Opti-MEM, TXNIP knockdown was induced by transfection of siRNA at a concentration of 100 nM. Following 48 hr of transfection, the cells were washed twice with PBS and then fixed with 100% ice-cold methanol for 10 min at –20°C. After rinsing three with PBSTw (PBS containing 0.1% Tween 20), the cells were blocked with 3% BSA in PBS and incubated for 45 min at room temperature. Next, cells were incubated with the primary antibody for 150 min followed by the secondary antibody for 60 min in the dark. For co-staining with a second primary antibody, the blocking step followed by the primary and secondary antibody incubation steps were repeated. All of the antibodies were diluted in antibody dilution buffer (1% BSA in PBS). Information of the antibodies is listed in the Key resources table. The cover slips were rinsed three times with PBSTw and then mounted with VECTASHIELD Antifade Mounting Medium containing DAPI (Vector Laboratories) according to the manufacturer’s instructions. The fluorescence was visualized with a Nikon C2 Si-plus confocal microscope. Fluorescence images were observed under a ZEISS confocal microscope (LSM5; Carl Zeiss, Jena, Germany), and the integrated densities of fluorescence were analyzed using ImageJ program.

Larvae were vortexed with glass beads for one minute before bleeding to detach sessile hemocytes. Next, the larvae were bled on a slide glass (Immuno-Cell Int. cat# 2018A29) and hemocytes allowed to settle onto the slide at 4°C for 40 min. Hemocytes were washed 3 times in 0.4% Triton X-100 in 1x PBS for 10 min and blocked in 1% BSA/0.4% TritonX in 1xPBS for 30 min. Primary antibody was added, and samples incubated overnight at 4°C. Hemocytes were washed 3 times in 0.4% Triton X in 1xPBS and then incubated with a secondary antibody with 1% BSA/0.4% Triton X in 1xPBS for 3 hr at room temperature. After washing 3 times with 0.4% Triton X in 1xPBS, samples were kept in Vectashield (Vector Laboratory) with DAPI and imaged by a Nikon C2 Si-plus confocal microscope.