Cy3 goat anti mouse antibody

On day 28, rats were perfusion-fixed, and the brain was removed, fixed, coronally sectioned and stained with NeuN and isolectin-B4 (IB4), a reliable marker of activated microglia, especially following ischemic stroke, as described in detail elsewhere [19 (link),46 (link),47 (link),48 (link),49 (link)]. Briefly, the brain was removed from the skull and kept in paraformaldehyde for 24 h, transferred to 30% sucrose solution (0.1 M phosphate buffer saline (PBS), pH 7.4) for at least 3–4 days, and cut into 40-μm-thick coronal sections on a sliding microtome (Leica). Sections were collected from across the MCA territory, i.e., from the level of the forceps minor of the corpus callosum to the visual cortex and the superior colliculi according to Paxinos and Watson [50 ]. Sections were then incubated for 2 h at room temperature in PBS plus 5% normal goat serum and 0.3% Triton X-100 (all Sigma). Sections were stained with anti-NeuN antibody (Millipore Bioscience Research Reagents, 1:500) and biotinylated IB4 (Sigma, 1:500) overnight at 4 °C, washed, and incubated with goat anti-mouse-Cy3 antibody (Jackson ImmunoResearch, 1:150) for 2 h at room temperature. Sections were washed, mounted on gelatine-covered slides, dried for 15 min on a heating block (40 °C) and coverslipped using FluorSave reagent (Calbiochem).

Six week old female Balb/cJ, C57BL/6J or gp91^phox-/- mice (Jackson Laboratories) were infected via tail vein. WT, isotype controls (Rat IgG2a for 1A8 from Bio X cell) and RB6-8C5 (Bio X cell) treated mice received 1x10⁵ cfu, those receiving 1A8 received 2.5x10⁴ cfu and gp91^phox-/- mice received 500 cfu. Mice were treated with isotype, RB6-8C5 or 1A8 antibody (Bio X cell; 100 μg in 200 μL of PBS) via i.p. injection on day 2 post-infection. On day 5 post-infection mice were sacrificed via CO₂ inhalation followed by cervical dislocation. Neutropenia was confirmed by Wright staining of blood obtained by cardiac puncture. Organs were harvested and homogenized as described [12 (link)]. For some experiments, kidneys were bisected with a razor and half was processed for histology. Homogenates were stained with sDectin-1-Fc (17 μg/ml) then donkey anti-human IgG Cy3 (0.8 mg/ml) and Calcofluor White (25 ng/mL). Alternatively, homogenates were stained with anti-β-glucan antibody (Biosupplies, Inc., Australia; 1.7 mg/mL) then with goat anti-mouse Cy3 antibody (Jackson Immunoresearch; 3.8 mg/mL). Cells were visualized by optical sectioning fluorescence microscopy using a Zeiss Axiovision Vivotome microscope (Carl Zeiss Microscopy, LLC). Maximum projection images were quantified using Cellprofiler (www.cellprofiler.org) as described [12 (link)].