Lacv monoclonal antibody 8c2.2

Midguts and ovaries were dissected at various time points following challenge with an LACV-containing bloodmeal and fixed in 4% paraformaldehyde–phosphate buffer solution (PBS) (Gibco, ThermoFisher Scientific) at 4 °C for a minimum of 30 min. Tissues were permeabilized in PBS-T (PBS, 1% BSA, 0.2% Triton X-100) at room temperature (RT) for 1 h. Tissues were then incubated overnight at 4 °C in PBS-T containing LACV monoclonal antibody (8C2.2) (Invitrogen, ThermoFisher Scientific (Waltham, MA, USA)) at a 1:200 dilution. Samples were then washed three times for twenty minutes each with PBS-T, then incubated with fluorescent-labeled secondary antibody (Alexa Fluor 594, Abcam: ab150120, Cambridge, UK) at a dilution of 1:500 and counter-stained with Phalloidin (AlexaFluor 488, Invitrogen, ThermoFisher Scientific) at 1:100 dilution for 1 h at RT. Cell nuclei were stained with DAPI (Invitrogen) at 1 μg/mL for ten minutes at RT. Tissues were washed three times again with PBS and mounted on six-well slides using Fluoromount G mounting medium (Electron Microscopy Sciences, Hatfield, PA, USA). Tissues were imaged using an inverted spectral confocal microscope (TCP SP8 MP, Leica Microsystems, Wetzlar, Germany) at the Molecular Cytology Core of the University of Missouri.

Ae. aegypti HWE and Ae. albopictus LC females received an LACV-containing bloodmeal (BM-1) (titer: 1.3 × 10⁶ PFUs/mL) followed by a non-infectious bloodmeal (BM-2) at 10-days post-BM-1. Mosquito bodies (without legs and wings) were collected at 24 h post-BM-2 and fixed in 10% neutral-buffered formalin at 4 °C for a minimum of 24 h. Mosquito bodies were then embedded in paraffin and sectioned onto microscope slides for IHC staining. Slides were incubated with LACV monoclonal antibody (8C2.2) (Invitrogen) at a dilution of 1:200 and incubated with a Mouse Envision secondary antibody for 20 min. All IHC staining was performed by the MU Veterinary Medicine Diagnostic Laboratory. Slides were imaged using a Leica ICC50 W microscope equipped with a Wi-Fi-enabled camera.