Cuticle sensitivity to hypochlorite was tested as described24 (link), 29 (link) with modifications. In brief, individual animals (L4 larvae or young gravid adults) were transferred into a 10 μl drop of freshly prepared 2%(v/v) alkaline hypochlorite solution (4 ml 5% sodium hypochlorite, 2.5 ml 4 N NaOH, 3.5 ml dH2O) on parafilm and the time taken until first visible rupture was noted.
Cuticle permeability to acridine orange (AO) and 4′,6-diamidino-2-phenylindole (DAPI) was assayed as described19 , 26 (link), 31 (link), 34 (link) with modifications. L4 larvae were washed from plates with M9 buffer prior to staining with AO or DAPI (5 μg/ml each in M9 buffer) for 15 minutes at room temperature with gentle agitation. Subsequently, worms were washed three times with M9 buffer, followed by fluorescence imaging. For microscopy, worms were mounted onto 3%(w/v) agarose pads, anaesthetised with 10 μl of 1 mM sodium azide and sealed with a coverslip before imaging on a Zeiss Axioplan 2 microscope. Samples were observed with a Zeiss Plan Neofluar 20x/0.50 Ph2 objective, images captured using a Zeiss AxioCam and the software AxioVision 4.8. AO or DAPI accumulation was imaged at 100msec exposure time, except for the strains CB7431 [bus-17(br2)] and AW1452 [bus-17(e2800)], where images were taken at 50msec exposure time.
Cuticle permeability to acridine orange (AO) and 4′,6-diamidino-2-phenylindole (DAPI) was assayed as described19 , 26 (link), 31 (link), 34 (link) with modifications. L4 larvae were washed from plates with M9 buffer prior to staining with AO or DAPI (5 μg/ml each in M9 buffer) for 15 minutes at room temperature with gentle agitation. Subsequently, worms were washed three times with M9 buffer, followed by fluorescence imaging. For microscopy, worms were mounted onto 3%(w/v) agarose pads, anaesthetised with 10 μl of 1 mM sodium azide and sealed with a coverslip before imaging on a Zeiss Axioplan 2 microscope. Samples were observed with a Zeiss Plan Neofluar 20x/0.50 Ph2 objective, images captured using a Zeiss AxioCam and the software AxioVision 4.8. AO or DAPI accumulation was imaged at 100msec exposure time, except for the strains CB7431 [bus-17(br2)] and AW1452 [bus-17(e2800)], where images were taken at 50msec exposure time.
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