Inverted confocal microscope

iPSCs or iRPE cells were plated onto vitronectin-coated coverslips in 24-well plates. Confluent cells (3–5 days for iPSCs, and a minimum of 42 days for iRPE cells) were fixed in 4% paraformaldehyde for 15 min, washed in PBS and incubated at room temperature for 1 h with a permeabilizing blocking buffer composed of 0.5% bovine serum albumin (BSA) and 0.05% saponin in PBS. Primary antibodies (Table S2) were incubated over night at 4 °C in permeabilizing blocking buffer, and washed three times with PBS, before incubation with the appropriate secondary antibody conjugated either with AlexaFluor 488 or 594 (Life Technologies, Thermo-Fisher Scientific) and with Hoechst 33342 (Invitrogen) in the blocking buffer for 1 h at room temperature. Coverslips were washed with PBS and mounted using Fluoromount (Invitrogen), and imaged with an inverted confocal microscope (Olympus, Tokyo, Japan).

FRAP was performed using the FRAP module on Olympus Confocal microscope Software. For FRAP, the amoebic trophozoites harbouring GFPEhC2Bwere observed 48 hours after addition of 20 μg/ml G418 using Olympus inverted confocal microscope equipped with 60x/1.4NA oil immersion objective and a 488 nm Mercury Arc laser. Bleaching was performed during fly forward using ROI scan features and high laser power. In the FRAP experiments, spherical areas of approximately 5 μm diameter were photobleached for 500msec, and subsequently, images of the area were collected approximately every 4 frames/s. To calculate the time of 50% signal recovery, τ_1⁄2, the FRAP curve from each of the experiments was normalised and fitted to an exponential recovery curve. The curve fit was done with help from CellSens software for the Olympus microscope.

Immunohistochemistry was performed on whole eye sections of paraffin-embedded or cryostat sections, as previously described (Sims et al., 2012 (link); Echevarria et al., 2013 (link); Duncan et al., 2017 (link)). To quench autofluorescence, tissue samples were treated with 0.1% sodium borohydride (Fisher Scientific), blocked in a solution containing 5% normal horse serum (NHS; Life Technologies) and 0.1% Triton X-100 (Fisher Scientific). Tissue was incubated overnight at 4°C in a solution containing the primary antibody (Table 1), 3% NHS, and 0.1% Triton X-100 in PBS, followed by a 2-h incubation with the appropriate secondary antibody solution containing 1:200 secondary antibody (donkey anti-mouse, -goat, -rabbit, and -guinea pig; Jackson Immuno, West Grove, PA, United States). Samples were counterstained with DAPI (Life Technologies) and mounted with Fluoromount-G (Southern Biotech; Birmingham, AL, United States). Fluorescent images of retina sections were taken with an inverted epifluorescent microscope (Nikon Instruments) or inverted confocal microscope (Olympus) and analyzed using FluoView Imaging program (Olympus) and ImageJ (NIH). Antibody isotype controls were used to identify non-specific labeling.

Labeling of whole-mount retina and retinal cryo-sections were done as previously described [11 (link),16 (link)]. Primary antibodies used were gp130 (1:200; Cat# MAB4681, R&D Systems) and β-Tubulin III (TUJ1, 1:500; Cat#845501; BioLegend). Secondary antibodies were used at a concentration of 1:200 and consisted of donkey α-rat 488 (cat# 712-545-150; Jackson Immuno) or donkey α-rabbit 647 (cat# 711-606-152; Jackson Immuno). Z-stack images (0.5 μm/stack) were obtained on an inverted confocal microscope (Olympus) at the Vanderbilt University Cell Imaging Services Core and were analyzed with FV-10 ASW image analysis software (Olympus).

As described previously (Lin et al., 2017 (link)), the mitochondria were labeled with MitoTracker Red CMXRos, M7512 (Thermofisher Scientific, USA) for 3 h after the brain slices were treated with LGLO. After an extensive wash, the slices were placed in an airstream incubator at 37°C and imaged by an Olympus inverted confocal microscope using a 60 × 1.3 NA oil immersion objective with 512 × 512-pixel resolution (FV1200, Olympus).
Upon imaging, a total of 5 min with 15 s intervals were imaged for each experiment. The total live imaging time was restricted to 20 min to minimize phototoxic damage. The length, area, and diameter of the axonal mitochondria were measured by the ImageJ program (NIH, USA). The number and mean velocity of motile mitochondria were analyzed by kymographs. Stationary sites in this study were defined as CMXRos-positive profiles that were stationary during a 5-min period. To measure the size of the stationary mitochondria, a pair of image stacks, including all CMXRos-positive profiles of each axon, were obtained at the time periods 0 and 5 min.

Autophagy was examined by analyzing the formation of fluorescent puncta of autophagosomes in cells transfected with mRFP-GFP-LC3. Cells were cultured in 24-well plates and transfected with mRFP-GFP-LC3 lentivirus. At 48 h post-transfection, the cells were treated with 200 µM GA in the absence or presence of CQ. Fluorescence images of indicated cells were directly taken using an inverted confocal microscope (Olympus).