F view soft imaging system

Larval hemocytes were isolated from the offspring of the indicated crosses. Ex vivo bacterial phagocytosis assays were performed as described earlier [32] (link), with the following modifications: wandering third instar larvae were disinfected in 5% sodium hypochlorite solution for 2 min, washed 3 times in H₂O, and bled into 1 ml ice-cold Schneider's Drosophila medium (Sigma-Aldrich). Then, excess medium was removed, 3×10⁶ FITC-labeled bacteria were added and centrifuged briefly onto the cells, and the cells were allowed to phagocytose for 10 min at 25 °C. Plates were returned on ice, the cells were fixed with 2% glutaraldehyde at room temperature, and extracellular fluorescent particles were quenched with a trypan blue solution. Microscopy and imaging were performed using Olympus IX71 microscope with F-view soft imaging system and QCapture Pro 6.0 software.

Soma and axonal compartments in the microfluidic units were fixed at room temperature for 1 h in 4% formaldehyde solution in phosphate-buffered saline (PBS). They were washed twice with PBS and permeabilized with blocking solution (2% BSA, 0.5% Triton-X-100 and 1× PBS) at room temperature for 1 h. We incubated the neurons/axons on both compartments with primary antibodies against synaptophysin (1:250) and MAP2 (1:4000) at 4 °C overnight. The next day, both compartments were washed three times with PBS and incubated with the secondary antibodies anti-mouse Alexa Fluor 546 (1:500) and anti-rabbit Alexa Fluor 488 (1:500) at room temperature for 1 h. After washing three times with PBS, both compartments were incubated with DAPI (1 µg/mL) for nuclear counterstaining at room temperature for 10 min. Both compartments were washed again three times with PBS prior to fluorescence microscopy. An Olympus IX81 time-lapse microscope (Olympus Deutschland GmbH, Hamburg, Germany) with a 10× objective (0.3 NA Ph1) and camera F-View soft Imaging system was used at room temperature. Images were acquired with Cell^TM software (Olympus Deutschland GmbH) and further processed via ImageJ (see Section 2.10).