Apotome

Imaging of cultures under bright-field and fluorescence microscopy was performed using an inverted fluorescence microscope (Olympus IX70, Center Valley, PA and Zeiss apo tome Thornwood, NY) with 10X, 20X objectives, DIC Nomarski, EGFP, Cy3, and DAPI filters. Images acquired on the Olympus microscope were saved in TIFF format while for the Zeiss apo tome the ZVI format was used. ImageJ was employed to extract individual channels from the ZVI format and resave them in TIFF format. After processing involving multichannel generation, brightness-contrast (brightness +50–150, contrast 10–100), level adjustments (0–80, 1.4–4, 255), montage, and channel mixing were performed using AdobePhotoshop CS6 (64 bit, San Jose, CA) with post-processing consistent across comparative images.

CD3⁺, CD8⁺, CD4⁺, Th1, or CVB4-specific T cells were labeled with cell tracker green (Life Technologies, USA) following the manufacturer’s instructions. T cells at a concentration of 4 × 10⁵ were added to the upper compartment of the insert facing the basolateral side of HIBCPP cells following 24 h of infection with E-30 at MOI 0.7. To the bottom of the plates (apical cell side), 1000 μl of MAM ± CXCL12 (200 ng/ml; Preprotech, Germany) was added for control conditions, since CXCL12 has been demonstrated to be an important chemokine in the CSF also in the context of neuroinflammation [9 ]. At the end of the 4 h of migration, the layers of HIBCPP cells were rinsed two times in PBS, fixed in 3.7% formaldehyde and stored for further immunofluorescence. The number of migrated T cells was evaluated microscopically (Zeiss Apotome, Germany) via counting of cells present on the bottom of the well. Ten fields of views were taken randomly in the well with a Zeiss Apotome and Zen software (Carl Zeiss, Germany) using an X10/1.4 objective lens. Quantification of the number of T cells was performed using Image J software.

At the end of the migration assays, HIBCPP cells were rinsed in PBS (Gibco, Thermofischer USA), and fixed in 3.7% formaldehyde for 15 min at room temperature (RT). HIBCPP cells were further washed in PBS, and culture inserts were cut out and permeabilized for 20 min with 1% Triton-X-100 PBS in 1% BSA at RT. Blocking was performed by incubating the cells in 1% BSA/PBS solution for 15 min at RT. As primary antibody monoclonal mouse light diagnostics™ anti-PAN Enterovirus (1:250; Merck, Germany) was used overnight at 4 °C. Cells were washed in PBS and incubated with the secondary antibody Alexa Fluor 594 goat anti-mouse, and simultaneously with phalloidin Alexa Fluor 660 (1:250 Molecular Probes, USA) and 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI) (concentration 1:50,000) for 1 h at RT, respectively. Cells were washed in PBS and mounted with antifade reagent (Life Technologies, USA). The quantification of T cells associated with HIBCPP cell layer was performed by counting the number of T cells present in 10 fields of views, which were selected randomly. Images were taken with a Zeiss Apotome and Zen software (Carl Zeiss, Germany) using an X63/1.4 objective lens. For evaluation of the migration pathway of the T cells, z-stacks from cell layers were acquired using Zeiss Apotome and Zen software (Carl Zeiss, Germany) using X63/1.4 objective lens.

Post-hoc histology was performed to confirm the location of the silicon probe. Both silicon probe shanks were coated with DiI (#C7000, Thermo Fisher Scientific). After the recordings, mice were deeply anesthetized with isoflurane then cardiac perfused with phosphate-buffered saline (PBS) followed by 4% Paraformaldehyde (PFA; Sigma-Aldrich). Whole brains were carefully dissected and post-fixed into 4% PFA overnight and sliced using a vibratome (Leica VT1200). Coronal serial sections were made at a thickness of 70 μm in a 4 °C PBS solution, mounted on a slide glass, and cover slipped with VECTASHIELD (H-1400, Vector Laboratories). We used the Paxinos Mouse Brain Atlas (Franklin and Paxinos, 2008 ) for nomenclature of brain regions. Images were taken using a widefield microscope (Apotome, Ziess). Contrast, brightness, and pseudocolor were adjusted in Image J (Schneider et al., 2012 (link)). Images were tilted to align to illustration based on the atlas.

To quantify myocytes in cell cycle and mitosis, paraffin-fixed tissue sections (5 μm) were incubated with either anti-Ki67 (mouse monoclonal antibody, clone MIB-1, Dako) or anti-phospho-Histone H3 (rabbit polyclonal antibody, Upstate Biotech). Positive cells were visualized by Alexa Fluor 488 (Thermo Fisher). To quantify myocytes in apoptosis we used TUNEL (Chemicon Inc.) staining as previously described [34 (link)]. Myocyte nuclei were identified with cardiac Troponin I and DAPI nuclear staining. Image acquisition was performed with a multiwavelength laser confocal microscope (Zeiss LSM 700) and AxioImager equipped with ApoTome (Zeiss) as previously described [28 (link), 37 (link)].