Eclipse c1si

Cells (5 × 10⁴ cells/well) were seeded for 24 h in 24-wells plates, exposed to FLG or GOs (10 μg/mL) for 72 h after probing cell membranes with 1 μM DiL as described above. Images were taken by a confocal microscope (Eclipse C1si, on an inverted microscope TE2000U, Nikon) at 60x magnification. FLG and GOs were visualized by the reflection mode property during the confocal acquisitions. Reconstructions of the images were performed offline using the image-processing package Fiji.

Immunofluorescent staining of retinal sections was performed with primary antibodies listed in (Additional file 2: Table S2) using our published protocols [38 (link), 51 (link)]. Anti-mouse or rabbit secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568 (Thermo Fisher Scientific, Waltham, MA) were used and counterstained with 49,69-diamidino-2-phenylindole (DAPI). Images were taken with a confocal microscope (Eclipse C1si; Nikon Instruments, Inc., Melville, NY). For non-fluorescent immunohistochemistry, primary antibodies were incubated overnight, then biotinylated mouse and rabbit secondary antibodies were incubated for one hour at room temperature. After incubation with Avidin/biotin complex (ABC) solution for one hour, retinal sections were incubated with nickel-intensified diaminobenzidine (DAB). Retinal sections were counterstained with CV, dehydrated in alcohols and cleared in xylene before mounting. Images were taken with a light microscope. In the GLP tumor/ toxicology study, tissue was collected by Absorption Systems (San Diego, CA) and processed by Inotiv (formerly, Seventh Wave, Missouri). For Yucatan minipig retinal sections, human-specific nuclear marker and nestin were used to identify donor cells.

tsA-201 cells were plated onto poly-d-lysine coated 35 mm glass bottom culture dishes (World Precision Instruments, Sarasota, FL)^{56 (link)}. Cells were transiently transfected with GFP-Cav3.1, GFP-Kv4.3, FMRP(1–297)-mKate or FMRP-mKate constructs to use as donor–acceptor fluorescent pairs^{56 (link)}. On the experimental day DMEM was replaced with imaging medium comprised of (mM): 148 NaCl, 3 KCl, 10 HEPES, 3 CaCl₂, 10 d-Glucose, 1 MgCl₂ (pH 7.3 with NaOH) at 25 °C. Cells were examined on a Nikon Eclipse C1Si spectral confocal laser-scanning microscope using a 40×/1.3NA oil immersion objective. Laser lines of 457 nm were used to excite GFP and 561 nm to excite mKate, with emission spectra recorded between 400 and 750 nm. Spectral images were linearly unmixed offline using ImageTrak software^{56 (link)}.

Confocal microscopy images of fixed samples were collected using EZ-C1 acquisition software on a Nikon Eclipse C1si confocal microscope. Images were analyzed and processed using ImageJ Fiji² (Schindelin et al., 2012 (link)). Wide-field live images were opened in ImageJ Fiji, and channels were individually bleach-corrected (simple ratio, background intensity = 0). Fluorescent intensity time courses were obtained for an ROI (region of interest) traced around the entire NMJ, or axon segment, in each frame in a given data set using Time Series Analyzer V3 plugin, averaging the fluorescence in the ROI. The time courses were saved as txt files and fed into R scripts written by the authors to obtain the resting fluorescence data (averaged 30 frames before stimulus) and the ΔF/F data (Supplementary Datasheet 3). Graphs were generated using GraphPad Prism 8. Figures were made using Adobe Illustrator.

SH-SY5Y cells (3 × 10⁵) and NSC-34 (3.5 × 10⁵) were plated in 6-well plates containing coverslips. For SH-SY5Y treated with siRNA against hnRNPs and NSC-34 we plated the corresponding number of cells in 6-well plates containing coverslips coated with poly-L-lysine solution at a final concentration of 0.01% (w/v) in H20 (SigmaAldrich, St Louis, MO, USA). After 24 h, cells were washed three times with PBS, fixed in 3.2% paraformaldehyde in PBS for 1 h at room temperature and permeabilized by using 0.3% Triton in PBS for 5 min on ice. Cells were then blocked with 2% BSA/PBS for 20 min at room temperature and immunolabeled with 1:200 rabbit polyclonal antibody anti-hnRNP Q (SigmaAldrich, St Louis, MO, USA) or 1:200 rabbit polyclonal antibody anti-hnRNP R (Abcam, Cambridge, UK) in 2% BSA/PBS overnight at 4°C. Next day, cells were washed three times with PBS, incubated with 1:500 anti-rabbit Alexa-Fluor 488 (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature and coverslipped with Vectashield-DAPI mounting medium (Vector Laboratories, Burlingame, CA, USA). Each slide was analyzed at the microscopy facility of University of Trieste, using a Nikon Eclipse C1si confocal microscope system mounted on a Nikon TE-2000U inverted microscope with a 60X objective.