C2 confocal laser microscope

A blocking solution (containing 5% milk, 1% bovine serum albumin, and 0.03% Triton X-100 diluted in 1× PBS) was applied to the previously sectioned spinal cords. Anti-GAD67 primary mouse antibody (Cat#: MAB5406; 1:500 concentration in blocking buffer) and anti-NEUN antibody (Cat#: ABN78; 1:500 concentration in blocking buffer) were applied overnight at 4 °C (Supplementary Table S1). The fluorophore-conjugated secondary antibodies (Cat#: A-21235 and A-11034; 1:500 concentration in blocking buffer) were applied for 90 min at room temperature (Supplementary Table S1). A 1× PBS solution was used to wash the cords three times during the staining process to minimize noise. The slides were imaged using a Nikon C2+ confocal laser microscope. The GAD67 images were quantified by image thresholding in Fiji, and the % area of positive GAD67 staining was recorded [50 (link)]. All images were analyzed in Fiji by a blinded examiner.

Lectins labeled by Cy3 fluorescent dye were applied to detect the specific sugar structure in cells according to our protocol [20 (link)]. Cells (2 × 10⁵) were seeded in 6-well culture plates containing sterile coverslips. The treated adherent cells were fixed with 4% paraformaldehyde and permeabilized in ice-cold 1× PBS supplemented with 1% Triton X-100 at 4 °C for 10 min and rinsed twice in 1× PBS. Prior to incubation with the labeled lectin, the fixed cells were blocked with the buffer (1× PBS supplemented with 4% BSA) at 37 °C for 30 min. The cells were incubated with the labeled lectin diluted to a final concentration of 15–20 μg/mL with the blocking buffer for 3 h at room temperature in the dark. Then, they were further stained with 1 μg/mL of DAPI (4′,6-diamidino-2-phenylindole) (Roche, Basel, CH) in 1× PBS for 10 min and a final rinse was performed. Nikon C2 Confocal Laser Microscope (Nikon, Tokyo, Japan) was used to collect the images with the merge channels of Cy3 and DAPI.

3-dimensional PAH media tissues were washed once with PBS, fixed with 4% (w/v) PFA in PBS (10 min, RT), permeabilized with 0.2% (v/v) Triton X-100 in PBS (5 min, RT), and blocked with Blocking One (nacalai tesque, Kyoto, Japan; >2 h, RT). The 3D-PAH media tissues were then incubated overnight at 4°C with Anti-Ki67 antibody (ab15580, rabbit polyclonal, Abcam, Cambridge, United Kingdom; final 1 μg/mL) diluted in Blocking One. After washing with PBS, 3D-PAH media tissues were incubated with Alexa Fluor 594-labeled donkey anti-rabbit IgG (H + L) secondary antibody (A-21207, Molecular Probes/Thermo Fisher Scientific; dilution: 1/200) diluted in Blocking One (30 min, RT). Finally, nuclei were stained with SYTOX Green (0.2 μM, 15 min, RT). Samples were observed under a Nikon C2+ confocal laser microscope. To quantify Ki67-positive cell nuclei, the area of Ki67-positive nuclei was divided by total nuclei area for four randomly chosen visual fields using ImageJ (National Institute of Health, Bethesda, MD, United States).

PKH67-labeled b.End5 cells were seeded at 4.0 × 10⁴ cells and allowed to adhere to 24-well plates for 4 h at 37°C under 5% CO₂. Then, 1.0 × 10⁴ cells of Mφ (–), Mφ (IL-10), Mφ (IL-18), or Mφ (IL-10 + IL-18) were overlaid onto fluorescent b.End5 cells and cocultured for 16 h at 37°C under 5% CO₂. Subsequently, photomicrographs were taken at 2–5 µm intervals for z-axis obtained at magnification 100× with a confocal laser C2 microscope (Nikon). The area of PKH67-positive region was calculated with NIS-Elements Ar software (Nikon) and evaluated as the number of surviving b.End5 cells.

RAW264.7 cells were fluorescently labelled with PKH26 (red) (MINI26, Sigma-Aldrich) according to the manufacturer’s protocol. PKH26-labelled RAW264.7 cells seeded at 4.0 × 10⁵ cells/dish in 35-mm glass-bottom dishes (Matsunami Glass, Kishiwada, Japan) were allowed to attach for 1 h followed by treatment with 200 μg/ml Alexa Fluor 488-BSA, -AGE-2, or -AGE-3 for 10 min or 4 h. The fluorescence images of Alexa Fluor 488-AGEs and PKH26-labelled RAW264.7 cells were captured at 0.5–0.8-μm intervals for the z-axis at original magnification × 400 using a confocal laser C2 microscope (Nikon, Tokyo, Japan).
In another experiment, RAW264.7 cells seeded at 4.0 × 10⁵ cells/dish in 35-mm glass-bottom dishes were allowed to attach for 1 h followed by treatment with 200 μg/ml Alexa Fluor 488-BSA, -AGE-2, or -AGE-3 for 4 h with or without of fucoidan (500 μg/ml). The fluorescence images of Alexa Fluor 488-AGEs were captured as described above.

RAW264.7 cells seeded at 4.0 × 10⁵ cells/dish in 35-mm glass-bottom dishes (Matsunami Glass, Kishiwada, Japan) were allowed to attach for 1 h followed by treatment with Alexa Fluor 488-labelled LPS and AGE-3 at 200 μg/ml for 4 h. The fluorescence images of Alexa Fluor 488-labelled LPS were captured at an original magnification of ×400 using a confocal laser C2 microscope (Nikon, Tokyo, Japan).