Biotinylated goat anti mouse

For immunohistochemistry experiments, tissues were stained with primary antibodies directed to identify microglia (rabbit anti-Iba-1, 1:4000, Wako or mouse anti-Iba-1 kindly provided by Dr. Bruce Trapp), inflammatory monocytes (rat anti-CD45, 1:2000, Bio-Rad), cerebellar neurons (rabbit anti-calbindin, 1:1000, Cell signaling Technology), pro-inflammatory cytokine interleukin-1 beta (rabbit anti-IL1 beta, 1:200, abcam), myelin basic protein (MBP) (rabbit anti-MBP, 1:2000, Invitrogen), and astrocytes (rat anti-GFAP, 1:4000, Invitrogen). Secondary antibodies were purchased from Jackson ImmunoResearch Laboratory as follows: Cy3-goat anti rabbit, Cy5-donkey anti rat, Cy2-goat anti mouse, biotinylated-goat anti rabbit, biotinylated-goat anti mouse, and biotinylated-rabbit anti rat. Biotinylated antibodies are detected by using stable DAB (Thermo Fisher Scientific) containing diaminobenzidine and hydrogen peroxidase.

In order to block the endogenous peroxidase and prevent non-specific binding, sections were first incubated in 3% hydrogen peroxide (VWR) in methanol for 30 min, followed by a 1 h incubation with 5% bovine serum albumin (Sigma) and 20% normal goat serum (Sigma) in 0.05 M TBS at room temperature. The sections were then incubated with the primary antibody (mouse anti-MeCP2, Sigma, 1: 250) overnight at 4 °C, rinsed with 1% TBS/Tween-20 and incubated with the secondary antibody (biotinylated goat anti-mouse, Jackson, 1:200) for 1 h at room temperature. Sections were then treated with Avidin-Biotin-Complex (Vectastain ABC kit, Elite-PK-6100 Vector Labs) for 30 min followed by incubation with 3, 3-diaminobenzidine (DAB) (Vector Laboratories). Finally, sections were stained with Mayer’s hematoxylin before being dehydrated and mounted with DPX. Images were captured using a CCD camera (Axiocam HRc, Zeiss, Germany) mounted on the light microscope (Eclipse 800, Nikon, Japan).

For immunohistochemistry experiments, tissues were stained with primary antibodies directed against Ionized Calcium-Binding Adapter Molecule 1 (IBA-1; as a microglia specific marker, 1:4,000; rabbit, Wako), IL-1β (1:200; rabbit, Abcam), glial fibrillary acidic protein (GFAP as an astrocytic marker, 1:4,000; Clone 2.2B10; rat, Invitrogen), NeuN (1:4000; clone: A60; mouse, Millipore), and Neuronal Class III β-Tubulin (1:1,000; clone: TUJ1; mouse, Covance). Secondary antibodies were purchased from the Jackson Laboratory and were as follows: Cy3-goat anti-rabbit, Cy5-donkey anti-rat, biotinylated-goat anti-mouse, biotinylated-goat anti-rabbit, and biotinylated antibodies were detected by using Cy3- or Cy5-conjugated streptavidin.