Immunofluorescence

Animals (n = 3 per experimental group) were anesthetized with isoflurane and perfused through the heart with heparinized (4,000 U/mL) saline (0.9% NaCl) followed by 4% paraformaldehyde in phosphate buffer (PB, 0.1 M; pH 7.4). Brains were dissected, post-fixed for 2 h, and cryoprotected in 30% sucrose in PB solution at 4°C. Forty micrometer-thick coronal brain sections, at the level of the middle cerebral artery territory (1.18 to −0.10 from Bregma), were obtained using a cryostat and collected in PB.

After a pre-incubation for 1 h in blocking solution (5% normal donkey serum, 0.3% Triton X-100 in PB), sections were incubated overnight at 4°C with the following primary antibodies: rat anti-MAC-1 (CD11b, 1:200 dilution, code MCA74; AbD Serotec), rabbit anti-myeloperoxidase (MPO, 1:200, code sc-16129; Santa Cruz Biotechnology), and/or rabbit anti-Ym1 (1:100, code 01404; StemCell Technologies) to label alternatively activated microglia/macrophages.^28,29 After three washes in PB, sections were incubated for 2 h at room temperature in a solution containing an appropriate mixture of the corresponding secondary antibodies labeled with AlexaFluor 488 or AlexaFluor 594 (1:400 dilution; Molecular Probes, Invitrogen). Finally, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 1:500; Sigma-Aldrich), and the sections were mounted on slides and coverslipped as previously described.³⁰ Immunostaining was examined under a fluorescence microscope (Leica DMI6000B; Leica Microsystems Srl) equipped with a high-resolution digital camera (Leica DFC350FX) and a dedicated software (LAS AF6000) for image analysis and deconvolution.

To quantify microglia/macrophages, MAC-1-immunopositive cells were counted in the ipsilateral striatum of vehicle- and azithromycin-treated mice subjected to transient MCAo. Briefly, three coronal brain sections, corresponding to the middle cerebral artery territory, were taken from each brain at 0.98, 0.38, and −0.22 mm from Bregma. Digitized images were acquired under identical microscope settings and cells were counted off-line, using ImageJ software, in three different optic fields of the confocal images (acquired through the 20 × objective). For each optic field, the total number of DAPI-stained cells labeled for MAC-1 were counted and expressed as number of immunopositive cells/mm².