Sp5 2 confocal microscope

Images were recorded using a Leica SP5 II confocal microscope equipped with a HCX PL APO 63x/1.40 NA LambdaBlue Oil immersion objective. Data were collected using 5× digital magnification at a 400 Hz/line scan speed (4 line average, bidirectional scanning) at 355 nm (3^rd harmonic NdYAG laser) with 3 mW laser power. In order to achieve excitation with maximal probe emission, the microscope was equipped with a triple channel imaging detector, comprising two conventional PMT systems and a HyD hybrid avalanche photodiode detector. The latter part of the detection system, when operated in the BrightRed mode, is capable of improving imaging sensitivity by 25%, reducing signal to noise by a factor of 5. Frame size was determined at 2048 × 2048 pixels, with a 0.6 airy disc unit determining the applied pinhole diameter rendering one voxel to correspond to 24.02 × 24.02 nm (frame size 49.16 × 49.16 μm) with a section thickness of 380 nm. This particular LSCM is equipped with a novel structural illumination module called PhMoNa (achievable resolution 62 × 62 × 280 nm).^{17 (link)} To detect sensitized Ln emission, a corresponding detection window of 400–800 nm was used to record images using only the above detailed 355 nm laser line.

Cells were grown on coverslips, fixed using 2.6% formaldehyde in PBS for 10 min, then permeabilized using 0.4% Triton X-100 in PBS for 5 min on ice. Cells were washed with PBS and dehydrated using an ethanol series (3 min in 70%, 80%, 95% and 100% EtOH). RNA probe, 2.5 μl, in 12 μl of hybridization buffer (1 ml 50% Denhardt’s solution, 200 μl of saline–sodium citrate (SSC) containing 20% dextran sulfate, 800 μl of formamide) was used for each coverslip of 22 × 22 mm². Probe was denatured at 75 °C for 8 min, then placed on ice for 2 min. Cells were inverted on to the probe on a slide, sealed with rubber cement and placed in a humid chamber at 37 °C overnight. After hybridization, the rubber cement was removed and cells washed 3× for 3 min with 2× SSC containing 50% formamide at 42 °C followed by 3× 3-min washes with 2× SSC at 42 °C, and briefly rinsed in water before mounting in DAPI-containing Vectashield. Images were acquired with a Leica SP5 II confocal microscope using LAS-AF software.

For immunostaining, neurons were cultured in poly-d-lysine-coated coverslips and treated with TNI, glutamate, and/or edonerpic maleate. After being fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and washed with PBS three times, neurons were blocked by 5% bovine serum albumin (BSA). Incubation with the DMPO (1:50, ab104902, Abcam) and Arc (1:50, sc-17839, Santa Cruz) primary antibodies was performed at 4 °C overnight. After being washed by PBS with Tween-20 (PBST) three times, the samples were incubated with the secondary antibody at 37 °C for 1 h. Then, incubation with 4′, 6-diamidino-2-phenylindole (DAPI) was performed to stain the nuclei, and the images were obtained using a Leica SP5 II confocal microscope.

For viability analysis, spheroids were stained using a Live/Dead cell-viability assay (Invitrogen) according to the manufacturer’s instructions (2 µM calcein AM and 4 µM EthD-1). Viability was quantified from confocal stacks (250 µm thick) acquired using a Leica SP5 II confocal microscope. The live cell area was reported as the ratio of calcein-AM stained area to the total spheroid area (sum of calcein-AM stained area and ethidium homodimer-1 stained area). Note, live cell area was used as a metric instead of per cell viability due to challenges in quantifying per cell viability in dense spheroids with the abundance of the cytosolic calcein signal making it difficult to visualize cell boundaries. As the calcein is a cytosolic signal and Ethd-1 is nuclear localized, the live cell area is an approximate heuristic for viability.