The analysis was performed as described [31 (link)]. Briefly, coverslips containing 100–200 cells/mm2 were fixed with (100%) methanol followed by treatment with cold ethanol and two rinses in phosphate-buffered saline (PBS). Samples were blocked with 3%bovine serum albumin (BSA) in PBS/Tween 20 (PBST) for 30 min and incubated in PBST containing 1%BSA, anti-IL-1Ra, and anti-GFAP antibodies. For dilutions of primary antibodies, please see Table 1 . After three washes in PBST (15 min each), slides were further incubated with Cy2 or Cy5 (Jackson ImmunoResearch). The samples were mounted and observed under an Olympus BX-41 fluorescence microscope.
Immunostaining of tissue was performed by fixing the brains in 4%paraformaldehyde followed by 30%sucrose [32 (link)]. Tissue was sealed into OCT and sectioned every 40 microns on a Leica Cryostat CM3050 S and kept in cryoprotectant. Frozen sections were treated with cold ethanol (–20°C), followed by two rinses in PBS, blocking with 2%BSA in PBST, and double-labeling with two primary antibodies (Table 1 ). After three washes in PBST, sections were further incubated with Cy2 and Cy5 (Jackson ImmunoResearcdh Laboratories). The samples were mounted and observed under an Olympus BX-41 fluorescence microscope. Counting analysis was performed using Olympus Microsuite V software with the help of a touch counting module.
Immunostaining of tissue was performed by fixing the brains in 4%paraformaldehyde followed by 30%sucrose [32 (link)]. Tissue was sealed into OCT and sectioned every 40 microns on a Leica Cryostat CM3050 S and kept in cryoprotectant. Frozen sections were treated with cold ethanol (–20°C), followed by two rinses in PBS, blocking with 2%BSA in PBST, and double-labeling with two primary antibodies (
