Icam 1

Brain sections were treated as previously described (Rocha-Ferreira et al., 2015 (link)). In brief, slides were fixed in 4% PFA, blocked with 5% goat serum (Sigma-Aldrich) for 30 min and incubated overnight with MAP2 (1:1000, Sigma), alphaM (1:5000, Serotec), CD68 (1:2000, Biorad), GFAP (1:6000, Dako) or ICAM1 (1:3000, Pharmingen) antibodies. Sections were then incubated with appropriate biotinylated secondary antibody (anti-rat or anti-rabbit; 1:100) for 1 h at room temperature, followed by 1 h incubation with ABC (1:100; Vector) solution and visualized with 3,3′ diaminobenzidene (DAB)/hydrogen peroxide (Sigma). The reaction was stopped and washed twice in bidistilled water. Slides were dehydrated by consecutive immersion in increasing concentrations of ethanol, isopropanol and xylene, then covered using DEPEX. Post-mortem neonatal sections were stained as previously shown (Carlsson et al., 2011 (link)). Slides underwent routine deparaffinization, followed by 15 min 1% hydrogen peroxide/PBS-Tween incubation before 15 min block with 5% goat serum. Overnight incubation with GLP1R (1:100, Novus Bio) was followed by 2 h room temperature incubation with biotinylated goat anti-rabbit secondary antibody (1:1000). Sections were incubated for 1 h with ABC (1:20) and visualized with DAB/hydrogen peroxide. The reaction was stopped, and sections covered as described above.

Flow cytometric analysis was performed in cells stained with the following Abs: CD90.2 [BioLegend; #105316; 1:300], VCAM-1 [BioLegend; #105712; 1:400], CD45 [BioLegend; #103116; 1:400], ICAM-1 [BD Biosciences; #553253; 1: 400], and CD11b [BD Biosciences; #557397; 1:400] using the FlowJo analysis software.

For surface-marker staining, cells were incubated with fluorochrome-conjugated Abs to CD4, CD8, CD11b, CD11c, CD80 CD86, VLA-4, ICAM-1, CD62L and CD44 (BD Biosciences, San Jose, CA) at the recommended dilution or isotype control Abs for 30 min on ice. To analyze MOG-specific Th1, Th2 and Th17 cells, splenocytes or CNS-infiltrating MNCs were stimulated with 25 μg/ml MOG peptide for 72 h or overnight, followed by stimulation with 50 ng/ml PMA and 500 ng/ml ionomycin in the presence of GolgiPlug for 5 h. Cells were surface-stained with mAbs against CD4 and CD8. Cells were then washed, fixed, and permeabilized with Fix & Perm Medium (Invitrogen), and intracellular cytokines were stained with Abs against IL17, IFN-γ, or IL4 (BD Biosciences). For phosphorylated STAT staining, cells were fixed with 4% paraformaldehyde for 10 min at 37 °C, permeabilized with 90% methanol for 30 min on ice, and stained with p-STAT1, p-STAT3, p-STAT4 and CD4 mAbs (BD Biosciences, San Jose, CA). Foxp3 staining was carried out using a commercial kit, according to the manufacturer’s instructions (eBioscience, San Diego, CA). Flow cytometric analysis was performed on FACSAria (BD Biosciences, San Jose, CA) and data were analyzed with FlowJo software (Treestar, Ashland, OR).

HUVECs (1 × 10⁶ cells per well) were seeded in a 60 mm dish and exposed to P. gingivalis LPS (5 µg/mL) alone, hispidulin alone (5 µM), or a combination of P. gingivalis LPS and hispidulin for 16 h. HUVECs were washed twice with PBS and incubated with phycoerythrin-conjugated anti-human CD54 (ICAM-1, 50 μg/mL, #555511, BD Biosciences, Bedford, MA, USA) at 4 °C. After 1 h, the cells were rinsed twice with 5 mL PBS and suspended in 500 µL PBS. The cells transferred into the FACS tube were analyzed using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

For cytofluorimetric analysis adherent cells were trypsinized, then washed twice in PBS, incubated with 5 μl of fluorochrome-conjugated antibodies for 30 min at 4 °C (5 × 10⁵cells/FACS tube), fixed with 1% formaldehyde solutions for 5 min at 4 °C, centrifuged for 5 min at 1600 rpm and washed once with 1 ml of PBS for 5 min at 1600 rpm. CD133/2 (293C3) (Miltenyi Biotec, Bergisch Gladbach, Germany), HLA-I, NGF-R, ICAM-1, CD20, CXCR4, CD10, c-Kit antibodies (all from BD Biosciences, Franklin Lakes, NJ, USA) conjugated with different fluorescent dyes were used; the unconjugated antibodies Nestin (Novus Biologicals, Minneapolis, USA) and Melan-A/MART-1 (Santa Cruz Biotechnology, Dallas, TX, USA) were used in combination of the goat-anti-mouse IgG-FITC (BD Biosciences) as secondary antibody and used after cell permeabilization. For apoptosis analysis cells were trypsinized, washed in PBS, fixed with 70% ethanol for 45 min at 4 °C, washed in PBS and stained with propidium iodide (50 μg/ml diluted in PBS) and RNAase (250 μg/ml), then stored for at least 3 h at 4 °C before analysis. Flow cytometer analysis was performed by BD FACScan™ System using CellQuest Pro software on a minimum of 5000 events for each sample.