Frosted glass slides

For transfers, spleens from chronically T. cruzi-infected mice were homogenized with frosted glass slides (Fisher Scientific) in a hypotonic ammonium chloride RBC lysis buffer and washed in RPMI with 10% FBS. CD8⁺ T cells were negatively selected through magnetic sorting with a CD8a⁺ T cell isolation kit (Miltenyi). CD8⁺ cells were transferred i.v. into infection-matched congenic mice, and recipients were terminated at various days post transfer to analyze donor cell populations in recipient tissues.

Human schwann cells seeded onto poly-D-lysine coated coverslips were treated with gp120 or heat-inactivated gp120 (control) and washed twice with PBS, and fixed with 4% PFA (Electron Microscopy Sciences) for 30 min at RT. The cells were then permeabilized with 0.01% Tween-20, washed twice with PBS and blocked with 3% BSA + 1% normal goat serum for 90 min at 4°C. Incubations with primary antibodies were done overnight at 4°C followed by two washes with PBS and incubation with secondary antibody for 90 min at 4°C. Coverslips were then washed and mounted on frosted glass slides (Fisher Scientific) with ProLong Gold antifade with DAPI before imaging on a Zeiss LSM800 confocal microscope. Controls for immunostaining specificity included staining neurons with primary antibodies without fluorescence-conjugated secondary antibodies (background controls), and staining neurons with only secondary antibodies; these controls helped eliminate auto-fluorescence in each channel and bleed-through (crossover) between channels. For live cell imaging, hSCs on poly D-lysine coated 35 mm² glass bottom petridishes (MatTek, P35GC-0-10-C) were transduced with BacMam Lysosome GFP for 36 h prior to imaging. Time lapse imaging with z-stacks at 2 min intervals were captured using a Zeiss LSM800 confocal microscope. Images were processed using ZEISS ZEN and Imaris 9.2 software.

Human schwann cells were plated on to poly-D-lysine coated coverslips in 24-well plates at a density of 5000 per cm² and treated with either recombinant gp120 or heat-inactivated gp120 (as control) for 40 min. The plate was chilled on ice and washed twice with ice-cold PBS (containing 1 mM CaCl₂, 2 mM MgCl₂) and LAMP1 antibody CD107a was added in 0.1% BSA in PBS for 30 min on ice. Following three washes with ice-cold PBS, plasma membranes were co-stained with PKH26 (4 μL/mL) for 90 s on ice. The cells were then washed three times with PBS, fixed in 4% PFA for 3 min on ice, and stained with secondary antibody (0.1% BSA in PBS) for 30 min. Coverslips were then washed, and mounted on frosted glass slides (Fisher Scientific) with ProLong Gold antifade (Catalog # P36935, Thermo Fisher) before imaging on a Zeiss LSM800 Confocal Microscope.