Ab25375

To investigate the effects of blocking various molecules present in the vascular endothelium, antibodies used included the following: RB40.34 (rat IgG1, 30 μg per mouse; BD Biosciences, catalog no. 553742), a monoclonal antibody (mAb) against murine P-selectin and 9A9 (rat IgG1, 30 μg per mouse), a mAb against murine E-selectin (provided by K. Ley, La Jolla Institute for Allergy and Immunology) or alternatively a mAb against murine E-selectin (rat IgG1, clone 96419, 30 μg per mouse; R&D Systems no. MAB5751-100). A 1:1 combination of P- and E-selectin antibodies was also used. Likewise, rat mAbs YN1/1.7.4 directed against ICAM-1 (50 μg per mouse, Abcam no. ab25375), rat mAb MK2 directed against VCAM-1 (50 μg per mouse, Abcam no. ab19569), or a 1:1 combination of YN1 and MK2 were used. Antibodies were injected intravenously (E-selectin and P-selectin) or intraperitoneally (all other antibodies) for two consecutive days before injection of purified schizonts/merozoites.

After i.c.v. LPS injection, the mice were anesthetized and perfused with ice-cold PBS to clear blood-borne proteins. Next, the brain was homogenized in 1 ml of cold PBS on ice, and the homogenate was centrifuged (12,000 rpm, 5 min). Cells were digested with radioimmunoprecipitation assay (RIPA) lysis buffer (50 mmol/L Tris–HCl, 150 mmol/L NaCl, 1 % Nonidet-40, 0.5 % sodium deoxycholate, 1 mmol/L EDTA, 1 mmol/L PMSF) for 30 min on ice and centrifuged at 12,000 rpm for 15 min at 4 °C. The brain homogenates or cell lysate were diluted in PBS and loading buffer, boiled (100 °C, 10 min), loaded on a 10 % acrylamide–SDS gel, and transferred to a Protran nitrocellulose membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5 % dry milk in PBS for 2 h at room temperature, incubated in primary antibodies against P-selectin (ab178424, Abcam, Cambridge, USA), E-selectin (ab18981, Abcam),VCAM-1 (ab134047, Abcam), ICAM-1 (ab25375, Abcam), CXCR2 (ab14935, Abcam), albumin (ab19194, Abcam), and β-actin (Cell Signaling, Beverly, CA, USA) overnight at 4 °C, washed, incubated in species-appropriate HRP-conjugated secondary antibodies for 1–2 h at room temperature in the dark, and washed three times. Then, the membranes were subjected to immunodetection using enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA, USA).