A1 confocal scanner

Anesthetized mice were transcardially perfused with a fixative solution containing 4% paraformaldehyde in 80 mM PIPES (pH 6.8), 5 mM EGTA, and 2 mM MgCl₂. Eyes were enucleated and post-fixed in the same solution for two hours at room temperature. After fixation, dissected eyecups were embedded in 2.5% low-melt agarose (Precisionary) and cut by a Vibratome (VT1200S; Leica) into 100 µm thick slices as described previously^{52 (link)}. Agarose sections were blocked in PBS containing 5% donkey serum and 0.5% Triton X-100 for 1 h at room temperature before staining with primary antibody in blocking buffer overnight at 4 °C. After primary antibody staining, sections were washed three times in PBS and incubated with secondary antibody in blocking buffer overnight at 4 °C. Finally, sections were washed three times in PBS and nuclei were stained with 10 µg/ml Hoechst (H3569; Thermo Fisher Scientific) for 30 min at room temperature. Finally, sections were washed three times in PBS, and mounted onto slides with Immu-Mount (Thermo) and coverslipped. Images were taken with a confocal microscope (Eclipse 90i and A1 confocal scanner; Nikon) with a 60× objective (1.4 NA Plan Apochromat VC; Nikon) using Nikon NIS-Elements software. Image analysis and processing was performed with ImageJ.

Live cells were mounted on a 2% agar pad made with selective minimal media, covered with a #1.5 coverslip, and imaged using a 100×/1.45 Plan-Apo λ objective lens on a TiE Eclipse microscope with A1 confocal scanner and GaAsP detectors (Nikon). The IB was photobleached using the 488-nm laser and recovery of fluorescence was followed by collecting z-stacks using a stage-mounted piezoelectric focus drive (Mad City Labs) over a depth of 3.6 μm at 100-nm intervals, every 15 s for 10 min or every 2 min for 20 min. Focus was maintained using the Perfect Focus System (Nikon). Pinhole diameter was set to 0.6 Airy units; the predicted optical section for our imaging parameters is 0.30 μm and the XY resolution is 0.18 μm, using Nikon Elements Confocal software. ImageJ was used to quantify fluorescence recovery. Intensity measurements were performed on a maximum-intensity projection. Cytoplasmic intensity was corrected for background and was measured in an area of the cytoplasm that was distant from the IB and did not include the vacuole. Integrated IB intensity was calculated as the product of area and mean intensity. IB perimeter was defined by thresholding to 1.4 times the mean cytoplasmic intensity. Mean IB intensity was corrected for photobleaching as described below. 3D rendering of the IB was performed in NIS Elements using the Maximum Intensity algorithm and a Z-zoom of 75%.