Fitc labeled dextran molecules

Forty-eight hour post blood meal mosquito abdomens were fixed and sectioned as described above [66 (link)]. Samples were sectioned at 5 μm, stained with hematoxylin and eosin (H&E) (Huntz Enterprises Inc., China) and Periodic Acid Schiff (PAS) (Sigma-Aldrich, China) according to the manufacturer’s protocol. Slides were hard mounted using Canada balsam (ChemsWorth). Slides were viewed using bright field illumination on a Nikon ECLIPSE IVi microscope connected to a Nikon DIGITAL SIGHT DS-U3 digital camera. Four days post dsRNA treatment A. stephensi were fed with blood meal supplemented with 500 kDa FITC-labeled dextran molecules (2.5mg/ml blood)(Sigma) which were filtered using PD MiniTrap Sephadex G10 columns (GE Healthcare) as described [27 (link)]. Forty eight hours post-feeding, midguts were dissected and FITC signal observed using a Zeiss, LSM710 confocal microscope connected to a Nikon DIGITAL SIGHT DS-U3 digital camera. Expression of 4 PM genes was analyzed 24 hr and 48 hr post blood meal using primers targeting peritrophin1(ASTE010406), peritrophin14 (ASTE009456), 2 chitinases, herein named chitinaseA (ASTE005630) and chitinaseB (ASTE000328) (S1 Table).

The 10, 40, and 70 kDa fluorescein isothiocyanate
(FITC)-labeled dextran molecules (Sigma-Aldrich) were encapsulated
at a concentration of 47.62 μM in 60 μL of 2.5%, 5%, and
10% hydrogels cast in flat-bottomed 96-well plates and allowed to
gel for 60 min at 37 °C. Once gelled, wells were topped up with
240 μL of 30 mM HEPES buffer (pH 8). A total of 60 μL
of solution was transferred to black bottom 96-well plates, and the
absolute fluorescence was measured using a Promega GloMax Discover
microplate reader (excitation 475 nm, emission 500–550 nm,
peak emission measured). Measurements were made every hour for the
first 4 h and then at regular intervals thereafter. Data were tested
for significance using a one-way ANOVA with Tukey’s multiple
comparison correction at 2 h and 24 h. Experimental data were then
normalized to steady state fluorescence by first fitting an exponential
plateau function of the form f(x) = Y_m × e^–kx where k represents a growth rate constant and Y_m is the maximum fluorescence value (R² values all >0.92). Fluorescence measurements were then normalized
to the end point value to enable comparisons between experimental
data and modeling results. These data were then used to parametrize
the model for D_eff.