Animals were anesthetized with 1.5% isoflurane, and placed in a David Kopf stereotaxic apparatus (Kopf, Tujunga, CA). Burr holes centered at +0.14 mm anterior to the bregma and 2.0 mm lateral to the sagittal suture were made bilaterally on the skull. LPS (Escherichia coli, serotype 055:B5, Sigma-Aldrich) was dissolved in 5 μl sterile pyrogen-free saline and was delivered through a Hamilton syringe/needle (33 gauge) over 5 minutes and left for 5 minutes. The delivery of LPS was aimed at 1.2 mm deep from the cortical surface. In preliminary experiments, we tested 5, 20, 50, and 100 ng of LPS (n = 3–5 for each dose) and analyzed brain sections by staining with Isolectin B4 (40 ng/ml, Griffonia simplicifolia lectin I, Sigma Aldrich) at 3 days after LPS injection (Fig. 1). Isolectin B4 was visualized by Vectastain ABC kit (Vector Laboratories, Burlingame, CA) with 0.02% 3,3-diaminobenzidine (DAB) tetrahydrochloride and 0.003% hydrogen peroxide. The other hemisphere received 5 μl pyrogen-free saline in the same manner, as a sham injection. Five nanograms LPS did not show positive cells (data not shown) as seen
