Goat anti mouse igg magnetic beads

Goat anti-mouse IgG magnetic beads (New England BioLabs Inc., Ipswich, MA, USA) were mixed for 1 h at 4 °C under constant shaking. The beads (3.65 × 10⁸) in a volume of 10 μL were coated with 10 μg of either anti-Map 1A6E10 mAb or anti-Map pAb. For negative controls, immunomagnetic beads were coated with an identical quantity of either a monoclonal antibody or a mouse polyclonal antiserum of a non-related specificity: anti-N-6-methyl adenine.¹⁷ (link) After 1 h at 4 °C under constant shaking, each set of antibody-coated beads was separated for 10 min using a magnetic rack, washed three times in PBS, resuspended in 100 μL of PBS, and stored at 4 °C until further use.

Zebrafish were euthanized in fish water containing 300 µg/ml tricaine, washed 2 times with PBS, and transferred individually to a 2-ml screw-cap tube with an O-ring. To each tube, a 6.35-mm steel bead and 1 ml of PBS were added. Fish were homogenized by a 15-s pulse using a Mini-Beadbeater-16 (BioSpec Products). To isolate Cryptococcus cells for imaging of capsule and cell size, a monoclonal antibody (kindly provided by Arturo Casadevall) was labeled with goat anti-mouse IgG magnetic beads (New England Biolabs catalog no. S1431S) according to the manufacturer’s instructions. The magnetic beads coated with the 18B7 antibody were diluted such that few beads were observed per yeast cell. The magnetic beads were incubated with the fish lysate at room temperature for 5 min, and beads were separated to the side of the tube for 5 min. The lysate was removed, and beads were washed 3 times with 1 ml of PBS. The beads were resuspended in 20 µl of PBS. Samples were prepared with India ink staining and imaged on a Zeiss Axio Imager A1 microscope with an AxioCam MRM digital camera. Image analysis was performed using ImageJ software.