Evos fl auto 2 imaging

Fixed eyeballs and lacrimal glands were embedded in paraffin and cut into 5 μm sections using a microtome (Leica RM2245, Leica Biosystems, Heidelberg, Germany). The sections were deparaffinized, hydrated, and stained with hematoxylin and eosin (YD Diagnostics Co., Yongin, Korea) The stained slides were observed using the EVOS FL Auto 2 imaging system (Thermo Fisher Scientific, Waltham, MA, USA).

HaCaT keratinocytes were incubated for 2 h with different concentrations of samples. The cell culture media was withdrawn from the wells, once washed, and then filled with PBS. Wells except the control were exposed to UVB (50 mJ cm⁻²) by a UVP CL-1000L ultraviolet cross-linker (Upland, CA, USA). Immediately after the irradiation, PBS was withdrawn, once washed, and replaced with serum-free culture media. Intracellular ROS levels were measured after incubating for an hour (DCFH2-DA assay) and cell viability was measured by MTT assay after incubating for 24 h [1 (link)]. SHC4 that indicated superior antioxidant and cytoprotective effects were further verified by DCFH2-DA staining of cells and analysis by fluorescence microscopy (EVOS FL Auto 2 Imaging, ThermoFisher Scientific, CA, USA) and flow cytometry (CytoFLEX, Beckman Coulter, PA, USA) based on methods described in our previous publications [8 (link)].

Microscopy images were captured on an EVOS FL Auto 2 imaging system (ThermoFisher) with brightfield and fluorescence imaging using GFP (470 ± 22 nm excitation, 510 ± 42 nm emission) or RFP (531 ± 40 nm excitation, 593 ± 40 nm emission) filter cubes. All images were captured with the EVOS 4× objective (fluorite, PH, long-working distance, 0.13 numerical aperture/10.58 mm working distance). We refined standard aggregation analysis by adding a median size adjustment step, where cellular area and aggregate area were normalized to their respective median area values across the entire experiment (see Supplemental Information). Aggregates of FUS begin to form at 24 h post-transfection, and all images of cells were taken between 24 and 72 h post-transfection. For live cell imaging, cells were incubated in the EVOS environment chamber at 37 °C and 5% CO₂ with humidity. Images were captured every 30 min beginning 24 h after the start of transfection for time courses of 18 to 44 h as indicated. Before beginning the time course, culture plates were placed in the environment chamber for 1 h to acclimate before fine-tuning fields of view. Fluorescence per cell and number of aggregates per cell were quantitated using a semi-automated approach in ImageJ (see Supplementary Information).