Hoechst nuclear dye 33258

To demonstrate the ability of L3 and L4 A. suum to ingest fluorescent probes, two function-based fluorescent probes were used in feeding assays: 1) beta-ala,lys-AMCA (BAL-AMCA), 200 μM (BP0352, BioTrend, Zurich, Switzerland) (Meissner et al., 2004 (link)); and 2) DQ Green-BSA (D-12050, Molecular Probes, Eugene OR), 100 μg/mL (Vazquez and Colombo, 2009 (link)). L3 and L4 were cultured in the presence of these probes for 4 h prior to assessment of ingestion. Bisbenzimide (BB,; Hoechst nuclear dye 33258, Sigma-Aldrich, St. Louis MO) was used at 10 μg/mL and incubated with larvae for 4 h prior to assessment by fluorescent microscopy to document whole larva inventories of nuclei in cells and organs observable with this dye. Propidium iodide (PI, P4170, Sigma-Aldrich, St. Louis MO) was used at 100 μM, and incubated along with BB for 4 h prior to addition of experimental treatments. Preincubation was intended to maximize access of tissues to the fluorescent probes in presence of various treatments. For most experiments, larvae were then incubated with the two (BB and/or PI) fluorescent probes and the treatment for the duration of the experiment.

Prior to extracting nuclei, we assessed numbers of nuclei that comprise the A. suum intestine. This was accomplished by staining freshly dissected intestine with the non-vital stain bisbenzimide (Hoechst nuclear dye 33,258, Sigma-Aldrich, St. Louis MO) at 10 µg/mL in PBS for 30 min. Rinsed tissue placed on a glass slide. The intestine assumed a flattened shape, rather than round. It was then viewed using a Nikon Diaphot 300 inverted microscope equipped with epifluorescence capabilities (UV-2 A filter (blue) and photographed with a Nikon D5100 digital camera. Digital images were evaluated by counting the average number of nuclei contained within 50µM × 50µM areas of intestinal tissue, and extrapolating the cell counts to the full intestine.
To obtain single nuclei preparations, flash frozen A. suum intestinal tissue (∼ 50 mg) was homogenized in a Dounce homogenizer in 3 mL Lysis Buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.025% NP-40, and 0.04 U/µL RNasin (Promega)) and incubated on ice 15 min. The suspension was filtered through a 30 μm filter to remove debris and pelleted at 500 × g for 5 min at 4^oC. Nuclei were washed and filtered twice with Nuclei Wash (1% BSA in PBS with 0.2 U/µL RNasin (Promega)). The nuclei pellets were resuspended in Nuclei Wash at 1000 nuclei / µL and filtered through a 40 μm FlowMi Cell Strainer.