Apo tirf 100 1.49 oil objective

The cells not analyzed by flow cytometry were used to prepare slides for confocal microscopy. Briefly, the cells were fixed with 4% (v/v) paraformaldehyde (Electron Microscopy Sciences) in PBS for 10 min at room temperature and washed 3 times with PBS followed by centrifugation at 500 × g for 5 min at 4 °C and resuspension in 100 μL of PBS. The cells were spun onto Superfrost Plus Microscope slides (Fisher Scientific) using a Statspin Cytofuge Cytocentrifuge (BECKMAN COULTER) at 27 × g for 4 min. Cell membranes were first stained using Alexa Fluor 555-conjugated wheat germ agglutinin (WGA555, ThermoFisher Scientific) (1 : 1000 dilution with 5% (v/v) goat serum albumin (GSA) in PBS) for 45 min at room temperature and washed 3 times using PBS. The cell nucleus was then stained with DAPI (ThermoFisher Scientific) (1 : 7500 dilution in PBS) for 5 min at room temperature and washed 3 times using PBS. After the cells were air dried, 4 μL of Fluoroshield (Millipore Sigma) was applied to a circular cover glass with a diameter of 12 mm (Fisherbrand), which was then mounted onto each slide. Fluorescence images were obtained using a Nikon A1R Confocal microscope with an Apo TIRF 100×/1.49 oil objective (Nikon). Collected images were analyzed using NIS-Elements AR Analysis 5.30 software (Nikon).

Third-instar larval preparations (muscles 6/7) were filleted and fixed in 4% paraformaldehyde, washed, and incubated overnight at 4°C with primary antibodies. Secondary antibodies were applied at room temperature for 2 hr. The following primary antibodies were used: anti-Myc (9E10 Santa Cruz), anti-GFP 3E6 (1:500; mouse; Life Technologies), anti-NC82 (1:100; mouse; Developmental Studies Hybridoma Bank), anti-HA antibody (1:1000; Rabbit; cell signaling technology) and anti-DLG (1:10,000; rabbit). Alexa-conjugated secondary (488, 555) antibodies and Cy5-conjugated goat ant-HRP were used at 1:500 (Life Technologies; Molecular Probes). Larval preparations were mounted in Vectashield (Vector) and imaged with an Axiovert 200 (Zeiss) inverted microscope, a 100X Plan Apochromat objective (1.4 NA) and a cooled charge-coupled device camera (Coolsnap HQ, Roper). Slidebook 5.0 Intelligent Imaging Innovations (3I) software was used to capture, process and analyze images. Structured illumination microscopy imaging was performed using the N-SIM Nikon system, consisting of a Nikon Ti-E Microscope equipped with a Apo TIRF 100×/1.49 Oil objective and an Andor DU897 Camera.