Ix81 inverted fluorescence microscope

Agarose-embedded retinal cross sections were prepared as previously described (Lobanova et al., 2010 (link)), collected in 24-well plates, and incubated for 2 h with Alexa Fluor 594 conjugate of wheat germ agglutinin (Invitrogen) in PBS containing 0.1% Triton X-100. Sections were washed three times in PBS, mounted with Fluoromount G (Electron Microscopy Sciences) under glass coverslips, and visualized using a Nikon Eclipse 90i Confocal Microscope.
Plastic-embedded retinal cross sections (1 μm thick) were prepared as previously described (Sokolov et al., 2004 (link)) and stained with toluidine blue for light microscopy. Tiled images of whole retina cross sections were obtained using the Olympus IX-81 Inverted Fluorescence Microscope, and aligned and stitched using the Olympus cellSens Dimension software. The number of photoreceptor nuclei in representative segments of outer nuclear layer (ONL) was quantified as a quantitative measure of surviving photoreceptors. The number of nuclei in a 400 μm segment of the ONL, located at 1 mm from each side of the optic nerve, was counted by hand.

EPC, RTG2 and RTgill cells were seeded onto Poly-L-Lysine (0.01% w/v) coated coverslips (Sigma-Aldrich, St. Louis, MO, USA). Following indicated treatments, cells were fixed with 4% paraformaldehyde (BM-155; Boston Bioproducts, Ashland, MA, USA) for 15 min at room temperature and then permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature. Cells were then blocked with 3% BSA, 0.02% Tween in PBS for 1 h at room temperature and then incubated the indicated antibodies at a 1:300 dilution in 3% BSA in PBS overnight at 4 °C. Cells were then incubated with a secondary antibody at a 1:300 dilution in PBS (Alexa 488- or Alexa 647-conjugated anti-immunoglobulin antibody, Molecular Probes; Eugene, OR, USA) for 1 h at room temperature. Cell nuclei were stained with DAPI (17985–50; EMS, Hatfield, PA, USA). Cells were imaged on an Olympus IX81 inverted fluorescence microscope and the analysis and processing of images were performed using Stream View and ImageJ software [70 (link)]. Cells containing G3BP1 puncta (n > 5) were considered stress granule positive and counted. The percentage of cells forming stress granules was calculated from at least 100 cells from various fields. Data are representative of three independent experiments.

Immunofluorescence was performed as previously described [9 (link)]. Transfected cells were analyzed in a motorized Olympus IX-81 inverted-fluorescence microscope equipped with an XM10-monochrome-camera and narrow band-filter cubes for UV (DAPI), green (GFP) and red (Cy3) excitation. The cells were stained with a Cy3-conjugted goat anti-mouse IgG or fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG and nuclei were stained with DAPI (4’6-diamidino-2-phenylindole dihydrochloride).

Coverslips with spores were prepared as described in Chelius et al., 2019. After 12 h of growth in YGV media at 28 °C the first coverslip was removed from media. One drop of calcofluor white stain (18909, Sigma, Darmstadt, Germany) was added before mounting to a slide. The slide was viewed under an Olympus IX-81 inverted fluorescence microscope (Olympus, Tokyo, Japan) and 30 images were captured, for each of three biological replicates (90 images total). Each image contained at least one mycelium. Following this initial assessment, micafungin was added to media at a final concentration of 10 ng/ml. Each hour thereafter, a coverslip was removed, and images were taken until coverslips were overgrown, ∼10 h later. The images are analyzed using Image J software (53 (link)) for their projected area and the number of branches and number of septa are counted for each mycelium. The growth and branching rates are calculated as described in Chelius et al., 2019 and significance was calculated based on three biological replicates (student's t test, p < 0.05).

TIRF imaging and analysis were done as previously described^{32 (link)}. An Olympus IX-81 inverted fluorescence microscope with a ×100/1.45 NA objective was used. 488 and 561 nm laser lines were used to image cells transiently transfected with DCV marker NPY-mNG or NPY-mCherry and GFP, mcherry or mRFP tagged proteins of interest. The image was projected onto an Andor Ixon 897 EMCCD through a DualView (Photometrics) containing 525Q/50 and 605Q/55 bandpass emission filters. Images were acquired using the Andor IQ2 software. Yellow-green 100-nm beads were visible in both channels of the image splitter and were used to align the red and green images with a projective image transform. Images were taken at the exposure of 100 with 500 ms interval. Pixel size was 160 nm. All analysis was performed using custom MATLAB (MathWorks) scripts and ImageJ. An unbiased quantitative correlation-based image mapping approach was used as previously described to determine the degree of association between the red-labeled test protein and all visible NPY-labeled dense-core vesicles and randomized control images^{31 (link)}.

alamarBlue^Ⓡ assay (Invitrogen, USA) was carried out to assess the effect of GF and CR supplementation on the tenocytes’ metabolic activity at each timepoint, as per manufacturer's protocol. Briefly, at the end of each culture timepoint, tenocytes were washed with Hanks’ Balanced Salt solution (HBSS, Sigma Aldrich, Ireland) and then alamarBlue^Ⓡ solution (10% alamarBlue^Ⓡ in HBSS) was added. After 3 h of incubation at 37 °C and 5% CO₂, absorbance was measured at 550 nm and 595 nm using Varioskan Flash spectral scanning multimode reader (Thermo Scientific, UK). Cell metabolic activity was expressed in terms of% reduction of the alamarBlue^Ⓡ dye and normalized to the control group without GF and without CR.
2.5. Cell nuclei counting
Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, USA; see Section 2.7.). Fluorescent images were captured with an Olympus IX-81 inverted fluorescence microscope and images were further processed with ImageJ software. Nuclei were counted to obtain cell number per area at the different timepoints.