Prolong antifade reagent containing dapi

Mice were deeply anesthetized with isoflurane and perfused transcardially with 4% paraformaldehyde in 0.1 M sodium phosphate buffer. Brains were post-fixed for 24 hours at room temperature, washed in phosphate buffer saline (PBS) and sectioned (50 µm) coronally using a vibratome (Leica VT1000s). Brain sections were mounted on superfrost slides, dried and coverslipped with ProLong antifade reagent containing DAPI (Molecular Probes). Whole brain sections were imaged with an Olympus VS110 slide-scanning microscope. High-resolution images of regions of interest were subsequently acquired using an Olympus FV1000 confocal microscope (Harvard Neurobiology Imaging Facility). All confocal images were acquired in a single plane and processed using ImageJ.

Uninfected and Toxoplasma infected cells were fixed with 4% paraformaldehyde and incubated with 3% HSA (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 1 h at room temperature. After subsequent incubation with the monoclonal Ab M86 (Enzo LifeScience Inc., Farmingdale, NY, USA) diluted 1:50 in 3% HSA/PBS for 14 h at 4 °C, the secondary antibody Alexa Fluor^® 647 (red) goat anti-mouse IgM (Abcam, Cambridge, UK) was used at 2 µg/mL in 3% HSA/PBS for 1 h at room temperature. Stained samples were mounted in ProLong Antifade reagent containing DAPI (Molecular Probes, Eugene, OR, USA). The cell preparations were examined using a Zeiss LSM 800 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) with oil immersion objectives (×63).
Identification and relative expression of α-Gal epitopes in S. mansoni cercariae and A. fumigatus conidia was performed by immunofluorescence and flow cytometry following the previously described procedures [27 (link)]. The α-Gal-specific monoclonal antibody M86 (Enzo LifeScience Inc., Farmingdale, NY, USA) and FITC-labelled goat anti-mouse IgM (Abcam, Cambridge, UK) were used as primary and secondary antibodies, respectively.