Eclipse ti2 epifluorescence microscope

Infected cells were washed with 1X PBS containing Ca²⁺ and Mg²⁺, fixed in 4% PFA, washed with 1X PBS, and then mounted on glass slides using Mowiol 4–88 mounting media with DAPI. For neuron enrich primary culture characterization, cells were permeabilized with Triton X-100 in PBS. The cells were incubated with a blocking solution of PBS containing Triton X-100 and goat serum (GS). The cells were then incubated with primary antibodies rabbit anti-MAP2 (neuron marker), mouse anti-NFM (neuron marker), mouse anti-GFAP (astrocyte marker), at dilution 1:200 and mouse anti-H8H9 (same as anti-GalC, matured oligodendrocyte marker) at dilution 1:50 prepared in PBS/GS/Triton X-100 for 1 h. The antibodies used with their source and dilutions are tabulated in Table 1. The primary antibody labeled cells were then washed thrice with 1X PBS for 5 min and then incubated with respective secondary antibodies prepared in PBS/GS/Triton X-100 for 1 h, as specified in Table 1. The fluorescence-labeled cells were carefully washed with PBS carefully avoiding exposure to light and then mounted. The slides were then observed for EGFP fluorescence. Images were acquired with a Nikon eclipse Ti2 epifluorescence microscope with Nikon DS-Qi2 coupled camera and analyzed using ImageJ software.

Images of SH-SY5Y cells were obtained with a non-confocal fluorescence microscope (Leica DMI6000 B) using a 60×/1.4 NA oil immersion lens and the LAS X software platform (Leica). Images of hiPSCs and hiPSC-derived motor neurons were taken with a non-confocal Eclipse Ti-2 epifluorescence microscope (Nikon) using the NIS-Elements AR software (Ver 5.01) and either a 20×/dry or 60×/1.4 NA oil immersion lens. Confocal images of hiPSC-derived motor neurons and mouse spinal cord were obtained with a super-resolution VT-iSIM microscope (Nikon) using a 100×/1.49 NA oil immersion lens. Deconvolution was performed with the NIS-Elements AR software (Ver 5.01) using the Richardson/Lucy algorithm with 20 iterations. For printing, brightness and contrast of individual channels were linearly enhanced using the Fiji software^{69 (link)}.

Fluorescence imaging was performed using a Nikon Eclipse Ti2 epifluorescence microscope equipped with a CSU‐W1 spinning disk confocal module and an Andor 4.2 Zyla sCMOS camera. The system was controlled by NIS‐Elements AR 5.21.03 64‐bit software. Images were taken using the following Nikon objectives: CFI Plan Apo Lambda 4× (0.2 NA), CFI Plan Apo Lambda 10× (0.45 NA), CFI Apo LWD Lambda S 20×WI (0.95 NA), CFI Apo LWD Lambda S 40×WI (1.15 NA), CFI Plan Apo Lambda 60×Oil (1.4 NA). DAPI was excited with a 405 nm laser and imaged with a 450/50 emission filter, Alexa Flour 488 was excited with a 488 nm laser and imaged with a 525/40 emission filter. Alexa Fluor 546 was excited with a 561 nm laser and imaged with a 607/36 emission filter. Alexa Fluor 647 was excited with a 640 nm laser and imaged with a 685/40 emission filter. During imaging, the gels were placed in glass‐bottom customed plates with all excess liquid removed. Poly‐L‐lysin coating was recommended recycled imaging plates.

HeLa cells were grown to 80% confluency in 6-well plates and transfected with 300 ng pcDNA3.1-FLAG-RBM39 constructs using Lipofectamine2000 (Invitrogen). On the following day, 40,000 HeLa cells were re-seeded in 8-well chambers (Bioswisstec AG) and fixed after 24 h with 4% PFA for 30 min. After three washes with TBS, the cells were permeabilised and blocked using 1x TBS, 0.5% (v/v) Triton-X-100, 6% BSA at room temperature for 1 h. Ms anti-FLAG M2 antibodies (Sigma) were diluted 1:200 in TBS+/+ (1x TBS, 0.1% (v/v) Triton-X-100, 6% BSA) and incubated with the cells overnight at 4 °C. After 3 × 5 min washes with TBS+/+, the secondary antibody (Chicken anti-Mouse AF488, 1:500, Invitrogen) was diluted in TBS+/+ and bound to the primary antibody at 37 °C for 1.5 h followed by incubation at room temperature for 30 min. Then, the slides were washed 5 times with 1x TBS and mounted with Vectashield HardSet mounting medium containing DAPI (Vectorlabs). Images were acquired with a non-confocal fluorescence microscope (Leica, DMI6000 B) using the Leica Application Suite software (LAS-X) or with a non-confocal Eclipse Ti-2 epifluorescence microscope (Nikon) using the NIS-Elements AR software (Ver 5.01) using a 60x/1.4 NA oil immersion lens. For printing, brightness and contrast of the pictures were linearly enhanced.

To assess for polyphosphate accumulation, the strains were cultivated in CDM in crimp-top serum bottles (see section 6.2) and harvested in the exponential phase. Cells were fixed with 4% formaldehyde for 30 min at room temperature, washed twice with PBS buffer (pH 7.2 to 7.6), and stored at 4°C overnight. Before analysis, the cells were permeabilized with 0.3% Triton X-100 for 10 min. Subsequently, the cells were incubated with 50 μg mL⁻¹ 4,6-diamidino-2-phenylindole (DAPI) for 10 min. The samples were analyzed using an Eclipse Ti2 epifluorescence microscope (Nikon). DNA was visualized by excitation at 365 nm and emission at 460 nm. Polyphosphate granules were visualized by excitation at 469 nm and emission at 525 nm.