Rat anti mouse cd11b

Immunohistochemistry was performed in the same manner as described by Boutin et al. [31 (link)]. Sequential sections of tissue, adjacent to those used for autoradiography, were stained either for glial fibrillary acidic protein (GFAP) and CD11b antigen (Ox42) or for GFAP and neuron-specific nuclear protein (NeuN). Sections were fixed in 10% formalin (Sigma-Aldrich, USA) for 1 h, washed in 6 × 5 min PBS, and then incubated for 20 min in diluent (PBS with 5% normal horse serum and 0.1% Triton X-100). Sections were then incubated overnight at 4°C with primary antibodies in diluent (rabbit anti-cow GFAP, 1 : 1,000, DakoCytomation; mouse anti-rat CD11b, 1 : 1,000, Serotec; mouse anti-mouse NeuN, 1 : 1,000, Chemicon) and then washed in 3 × 5 min PBS. Incubation in secondary antibodies was for 1 h at RT (Alexa Fluor 594 nm goat anti-mouse IgG, 1 : 500; Alexa Fluor 488 nm goat anti-rabbit IgG, 1 : 500 (Molecular Probes, Invitrogen)) and then sections were washed again in 3 × 5 min PBS. Sections were mounted with an antiadherent solution (Citifluor, UK). Sections incubated without primary antibodies served as negative controls.
Immunofluorescence images were obtained with an Olympus BX-41 microscope equipped with a digital camera (QImaging, Canada) at 10x magnification, acquired with Image-Pro software. Image fusion was performed with MCID image analysis software.

Immunohistochemistry staining was performed at day 7 after reperfusion. The animal was terminally anesthetized and sacrificed by decapitation. The brain was removed, frozen, and cut into 5-μm-thick sections in a cryostat. Sections were fixed in acetone (− 20 °C) during 2 min, washed with phosphate-buffered saline (PBS) and saturated with a solution of bovine serum albumine (BSA) 5%/Tween 0.5% in PBS during 15 min at room temperature, and incubated during 1 h at room temperature with primary antibodies in a solution of BSA (5%)/Tween (0.5%) in PBS. The section was stained for CD11b with mouse anti-rat CD11b (1:300; Serotec, Raleigh, NC, USA) and for TSPO with a rabbit anti-rat TSPO (NP155, 1:1000). Sections were washed (3 × 10 min) in PBS and incubated for 1 h at room temperature with secondary antibodies Alexa Fluor 350 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG (Molecular Probes, Life Technologies, Madrid, Spain, 1:1000) in BSA 5%/Tween 0.5% in PBS, washed again (3 × 10 min) in PBS, and mounted with a prolong antifade kit in slices (Molecular Probes Life Technologies, Madrid). Standardized images acquisition was performed with an Axio Observer Z1 (Zeiss, Le Pecq, France) equipped with a motorized stage.