, Canada) and the exposed brain was kept moist with an artificial cerebrospinal fluid buffer, an ionic composition in mmol/L: NaCl 132, KCL 2.95, CaCL2 1.71, MgCL2 0.64, NaHCO3 24.6, dextrose 3.71 and urea 6.7, pH 7.4, at 37 °C. To observe leukocyte/endothelium interactions, leukocytes were fluorescently labeled by intravenous administration of rhodamine 6G (0.5 mg/kg body weight) and observed using a microscope (Olympus B201, ×20 objective Pictilisib cost lens, corresponding a 100 μm of area) outfitted with a fluorescent light source (epi-illumination at 510–560 nm, using a 590 nm emission filter). A
silicon-intensified camera (Optronics Engineering DEI-470) mounted on the microscope projected the image onto a monitor (Olympus). Rolling leukocytes were defined as white cells moving at a velocity less than that of erythrocytes cells. Leukocytes were considered adherent to the venular endothelium if they remained stationary for 30 s or longer. Brain tissue extracts were obtained from control and experimental mice that were sacrificed at 14 days after immunization. Brains PKC inhibitor were removed after intravital microscopy, and left and right hemispheres were stored on ice. Thereafter, frozen hemispheres were homogenized
in extraction solution (100 mg of tissue per 1 mL), containing 0.4 M NaCl, 0.05% Tween 20, 0.5% BSA, 0.1 mM Phenyl methyl sulphonyl fluoride, 0.1 mM benzethonium chloride, 10 mM EDTA and 20 KIU aprotinin, using Ultra-Turrax. Brain homogenate was spun at 12,500×g for 10 min at 4 °C and supernatants were collected and stored at − 70 °C. The concentration of MCP-1/CCL2 (Monocyte Chemotactic Protein 1/Chemokine C–C motif Ligand 2), RANTES/CCL5 (Regulated upon Activation, Normal T-cell Expressed, and Secreted/Chemokine C–C motif Ligand 5) and IL-17 was determined using
ELISA. After sacrifice, brain was removed from mice and leucocytes were then isolated from the brain by homogenization Leukotriene-A4 hydrolase through a nitex screen into RPMI (Roswell Park Memorial Institute) media. This cell suspension was fractionated using a step gradient consisting of 30% percoll (Sigma, St. Louis, MO) diluted in RPMI layered over 75% percoll diluted in RPMI. After centrifugation (8000×g), myelin was aspirated off the top of the 30% percol layer. Leucocytes were removed from the interface between the 75% and 30% layers of percoll. Afterwards, leucocytes were centrifuged (600×g) and resuspended in 1 mL of a solution containing 0.5% Bovine Serum Albumin (BSA), 2 mM azide and phosphate-buffered saline (pH 7.4). Leukocytes obtained from CNS tissue were stained with a combination of fluoresceine isothiocyanate (FITC) and phycoerythrin (PE) labeled antibodies directed against surface molecule CD4 and intracellular molecule IL-17, respectively. Data were acquired using a FACScan (Becton Dickinson, San José, CA, USA) and raw data of FACS analysis were processed using the Cell Quest software (Becton Dickinson, San José, CA, USA).