Fluoromount g mounting medium

Fixed coronal brain sections, neurospheres, or single NSC were permeabilized with 0.1% Triton X-100 for 10 min, blocked with 5% fetal bovine serum in 0.1% Triton X-100 for 1 h at room temperature, and then incubated with primary antibodies at 4 °C overnight. Incubation with the appropriate secondary antibodies or FITC-Phalloidin (1:200, Sigma) and DAPI (1:10,000, Molecular Probes, USA) was performed at room temperature for 1 h. Glass slides were mounted using Fluoromount G mounting medium (Electron Microscopy Sciences, USA). Brain images were captured on a Leica TCS SP8 confocal microscopy using LASAF software (Leica, Germany), and NSC images were captured on an Olympus FluoView 300 confocal system using the FluoView software (Olympus, Japan).
Primary antibodies: mouse anti-CSPG (1:250, Abcam, USA); guinea pig anti-DCX (1:1000, Millipore, USA); chicken anti-GFAP (1:500, Abcam). Secondary Antibodies (Invitrogen, USA): Alexa Fluor 594-conjugated goat anti-mouse IgG (1:300); Alexa Fluor 488-conjugated goat anti-guinea pig IgG (1:1000); Alexa Fluor 488-conjugated goat anti-chicken IgG (1:500).

Ae. aegypti larvae were pinned dorsal side up on sylgard dishes (Dow corning) containing ice-cold PBS, filleted along the dorsal midline, and pinned open to expose the VNC. Intestines, fat bodies, and trachea were removed, samples were rinsed in PBS, and subsequently fixed for 30 min in PBS + 4% formaldehyde (PBSFA) on a rotary shaker. Following fixation, samples were washed twice with PBS for 2 min each and subsequently permeabilized PBST (PBS + 0.3% Triton X-100) for 15 min (3 washes, 5 min each). Specimens were blocked for 15 min in PBST containing 5% NGS (Jackson Immunoresearch) and incubated in primary antibody diluted in blocking buffer at 4 °C overnight. The following day, samples were washed twice with PBS for 2 min each, three times with PBST for 10 min each, and incubated with the secondary antibody and Hoechst (1:500) at room temperature for 1–2 hours in blocking buffer. Following staining, samples were washed with 800 μl PBST 3 times for 10 min each at room temperature, rinsed twice in PBS, and mounted in Fluoromount G mounting medium (Electron Microscopy Sciences). Slides were dried at room temperature for a minimum of 1 hr. prior to imaging. Details on antibody sources and dilutions are provided in Table S1.

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.

Air dried bone marrow smears were hydrated with 1X PBS, fixed with 4% formaldehyde and permeabilized with 0.5% Triton X-100. Immunostaining was performed using a rabbit anti-FBL antibody at 1/500 dilution (ab5821, Abcam), a mouse anti-NCL antibody at 1/2000 ([4E2] ab 13541, Abcam) and AlexaFluor 488^®/AlexaFluor 555^® conjugated secondary antibody (Molecular Probes). Following immunostaining, nuclei were stained with Hoechst 33342 (Molecular Probes), and smears were mounted with Fluoromount G mounting medium (Electron Microscopy Sciences). Images were collected with a x63 NA 1.4 Plan-Apochromat objective on a Carl Zeiss LSM 780 confocal microscope. Image analysis and quantification were performed on wide-field images collected with a x40 NA 1.30 Plan Fluor objective on a Nikon NiE microscope equipped with an Orca Flash 4 camera (Hamamatsu), and using the NIS Elements software (Nikon). Briefly, nuclei were segmented using intensity thresholding of Hoechst signal, and saved as individual ROI (Region Of Interest). Nucleoli were segmented using intensity thresholding of FBL signal within the ROIs and their number and size were extracted.

Guts were dissected in PBS and transferred into 4% PFA immediately after dissection. After 20 min of fixation, guts were washed three times with 0.3% PBT. Stainingbuffer (0.15M NaCl; 1 mM MgCl₂; 10 mM Na-phosphate buffer pH 7.2; 3.3 mM F3Fe(CN)6, 3.3 mM K4Fe(CN)6; 0.3% Triton X-100) was heated to 65°C and 3% X-Gal added. The guts were stained for at least 1 hr at 37°C until a dark blue staining became visible. Guts were washed two times in 0.3% PBT prior to mounting in Fluoromount-G Mounting Medium (Electron Microscopy Sciences). Stained midguts were imaged using an Axiophot2 microscope (Carl Zeiss) equipped with an AxioCam MRc (Carl Zeiss).