Eclipse 2000

From tumors of each treatment group, 4-μm-thin tissue sections were cut using a microtome and transferred to gelatin-coated slides [36 (link)]. Tissue sections were incubated with primary antibodies anti-FADD (ab24533), anti-apoptotic protease activating factor 1 (APAF-1) (ab2001), and BCL-2 (ab32124; Abcam, Burlingame, CA, USA) at 4 °C overnight. Slices were washed with phosphate-buffered saline (PBS) and incubated with a streptavidin/Haptoglobin Related Protein (HRP)-conjugated secondary antibody (Biocare Medical, Concord, CA, USA). Immunoreactivity to the various proteins was visualized with a colorimetric-based detection kit following the protocol provided by the manufacturer (TrekAvidin-HRP Label + Kit from Biocare Medical, Pacheco, USA). Light microscopy (Nikon Eclipse 2000 equipped with Nikon DS-Fi2; Nikon Corporation, Tokyo, Japan) with a high-power objective (40×) was used to acquire digital images. The intensity of cell immunostaining was scored as follows: 1 = absence of positive cells, 2 = small number of positive cells or isolated cells, 3 = moderate number of positive cells, and 4 = large number of positive cells. Labelling intensity was evaluated by two previously trained examiners in a double-blind fashion.

The multilayer 3D model was made via SolidWorks 2015 and saved as a standard triangulation language (.stl) file, for the trajectory generation with Slic3r™ (https://slic3r.org/). Then, the aforementioned file was converted into g-code format, useful for the customized firmware (Marlin™) and, consequently, motion control. Hence, the geometry regularity of the OBST (variability between digital and printed model) was measured using an inverted epifluorescent microscope (Nikon, Eclipse 2000, Balsamo Strumenti, Medicina, Italy) and the pictures of the scaffold were acquired using a microscope camera (CCD Sony Corporation, Shinagawa, Tokyo, Japan). Finally, the expanded cells, included in the bioink, were seeded following the CAD drawing (from 7 to 21 crisscrossing sinusoidal 180° phase-shift layers (honeycomb)) resulting in a defined 3D assembly of 16 × 16 × 1.5 mm.

Images were obtained from fixed dissociated neurons on an inverted Nikon Diaphot microscope with a CoolSnap ES camera controlled by Metamorph software. Scoring for rods was performed blindly; randomized samples were not identified until all coverslips had been scored. Coded coverslips were scanned over several different regions and for most experiments100 neurons per coverslip were examined and scored as positive for rods if they contained a single rod. Rod-containing neurons interacting with other neurons were scored as one positive neuron since it was not possible to determine from which soma a rod containing process originated, whereas non-rod-containing neuronal networks were scored for each soma that they contained since none of the neurons within the network had rods. Triplicate or quadruplicate coverslips for each treatment were used in each experiment and experiments were repeated at least three times, giving between 800 and 1200 neurons scored for each treatment.
Live cell imaging was performed on a Nikon Eclipse 2000 inverted TIRF microscope with 405, 488, 561 and 640 nm laser lines, perfect focus control, XY piezo Z stage, CO₂-controlled stage incubator, 100X (1.48 NA) and 40X (0.75 NA) objectives and Andor iXon3 EMCCD camera. Images were captured and analyzed using Nikon Elements software.

For ICC experiments of VCP, 661W mouse retinal cells and human hTERT-RPE1 cells were cultured in a 24 well plate on 0.5% gelatin-coated glass coverslip. After siRNA transfection, the cells were washed once with Dulbecco’s phosphate-buffered saline (DPBS) and fixed in 4% paraformaldehyde for 10 min. Subsequently, the cells were washed twice with 1×DPBS and incubated for 1 h in blocking solution (0.1% Triton X-100, BSA 2% in 1×DPBS). After, immunofluorescence experiments were performed using a 1:100 dilution of mouse monoclonal anti-VCP (Table S1) in blocking solution and a 1:200 dilution of appropriate anti-mouse IgG Alexa Fluor™ 568 dye-conjugated antibodies (Table S1) in 1xDPBS. The nuclei were stained using DAPI (Roche, Switzerland) at 3.5 µM in 1×DPBS. Slides were mounted with Vectashield media for fluorescence microscopy (Vector laboratories, Peterborough, UK) and imaged under fluorescence microscopy (Nikon Eclipse 2000, Melville, NY, USA).

GEC phenotype was confirmed by the Periodic Acid-Schiff (PAS) staining in cell cultures collected on days 0, 7 and 15. 1 × 10⁴ cells were harvested at each time point, seeded onto microscope slides that had been previously coated with collagen type I and incubated at 37 °C, 5% CO₂ for 24 h. Then, GECs were fixed with a 4% paraformaldehyde solution in phosphate-buffered saline (PBS) for 15 min and washed in PBS. The slides were treated in a solution of 0.5% periodic acid for 5 min and stained with Schiff’s reagent for 15 min. After removing Schiff’s reagent, the slides were rinsed with running tap water for 10 min. Finally, cells were counterstained with a hematoxylin solution for 5 min. All steps were performed at room temperature (RT). A uniform reddish-purple cytoplasm was considered positive for PAS staining. Imaging was made using a Nikon Eclipse 2000 microscope.