Dextran feeding assays were performed by employing a modified version of previously described protocols (24 (link), 8 (link)). In brief, 500 kDa FITC (fluorescein isothiocyanate)-labeled dextran molecules (Sigma) were dissolved in a 2.5% sucrose solution and filtered using PD MiniTrap Sephadex G10 columns (GE Healthcare). Tsetse (n=10 individuals of each dsRNA treated group, with the exception of dsCS/LC) were inoculated with dextran by feeding flies a 2.5% sucrose solution containing 10% bovine blood and 10% filtered dextran molecules (1 mg/ml). Six hours post-feeding, midguts from dsGFP, dsP1/2 dsCS and dsCS/P treated individuals (n=3 per group) were dissected and FITC signal observed using a fluorescent dissecting microscope (Zeiss Discovery) equipped with a digital camera (Zeiss AxioCam MRc 5).
Pd minitrap sephadex g10 columns
Manufactured by GE Healthcare
The PD MiniTrap Sephadex G10 columns are disposable desalting and buffer exchange columns designed for the removal of low molecular weight compounds from protein samples. The columns use the size exclusion chromatography principle to separate the target protein from small molecules. With a fractionation range of 700-10,000 Da, the PD MiniTrap Sephadex G10 columns are suitable for purifying, concentrating, and desalting proteins and peptides.
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2 protocols using pd minitrap sephadex g10 columns
1
Dextran Feeding Assay in Tsetse Flies
2
Mosquito Midgut Histology and Permeability
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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 ).
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