Goat polyclonal anti iba 1 antibody

Mice were anesthetized by intraperitoneal injection of chloral hydrate (40 mg/kg), and transcardially perfused with ice-cold phosphate-buffered saline followed by 4 % para-formaldehyde. The brains were quickly taken out, immersed in 4 % para-formaldehyde for fixation at 4 °C overnight, and processed for paraffin embedding. Coronal sections (5 μm) of hippocampus were prepared for the histological examination. The sections were treated with 3 % H₂O₂ for 10 min, and then incubated in 5 % goat serum for 30 min. For Iba-1 staining, the sections were incubated with a goat polyclonal anti-Iba-1 antibody (Abcam, Cambridge, UK), and then incubated in biotin-labeled anti-goat IgG antibody (Bioworld Technology, Inc., St. Louis Park, MN, USA) for 2 h at room temperature. The immunoreactivity was visualized by the standard avidin-biotin complex reaction with 3, 3′-diaminobenzidine (Vector Laboratories, Burlingame, CA, USA). Iba-1 positive cells were counted using a light microscope (Olympus, Japan). The density of Iba-1 positive cells was expressed as the mean number of Iba-1 positive cells per mm².

Monoclonal antibodies against the catalytic subunit p110γ of PI3Kγ was produced in our laboratory [23 (link)]. Primary antibodies for phospho-CREB (#9198) and CREB (#9104) were purchased from Cell Signaling (Danvers, MA, USA). Goat polyclonal anti-Iba-1 antibody (#ab5076, Abcam, Cambridge, UK) was used for Iba1 staining. Antibodies against β-actin (#A2228 and #A5441) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Secondary HRP-coupled anti-rabbit and anti-mouse antibodies were purchased from KPL (Weden, Germany).

Tissue was prepared for labeling as described for stereological quantification of NeuN positive neurons above. Coronal sections spaced 360 µm apart throughout the striatum (+1.70 mm to −2.30 mm relative to Bregma) were collected and stained with goat polyclonal anti-Iba1 antibody (1∶600 dilution; Abcam, Cambridge, MA) using a free-floating protocol. Alexa Fluoro 594 goat anti-rabbit IgG (1∶1000 dilution; Invitrogen, Carlsbad, CA) was used as the secondary antibody. Cover slips were applied to mounted brain sections using Fluoro-Gel anti-fade (Electron Microscopy Sciences). Serial fluorescent images were captured and the number of striatal microglia per field (field size 832×666 µm, width x height, 1 field per section) was quantified by using Nikon NIS-Element software.