Phd Thesis 33 - eCommons@USASK - University of Saskatchewan

(Broehan et al., 2007), was also identified in M. configurata and its expression pattern was
examined.
3.2 Material and Methods
3.2.1 Microscopy
PMs were examined by epifluorescence, confocal, and scanning electron microscopy
(SEM). Epifluorescence microscopy was used to image the general physical architecture of the
PM at different conditions. PMs from feeding or starved (24 h without food) 5 th instar larvae and
molting Phase II larvae (5 th to 6 th instar) (molting phases were defined in Chapter 2) were
incubated in 0.5% (w/v) Fluorescent Brightener 28 (FB28) (Sigma, St Louis, MO, USA). The
PMs were rinsed in distilled water and photographed under brightfield or fluorescence
microscopy (Zeiss Axiocam) with a 0.8X objective (Neolumar S) using a digital camera
(Axiocam HRc).
Confocal microscopy was used to examine the PM in greater detail to investigate the
multilayered structure observed using FB28 staining with fluorescent miscroscopy. PMs from 4 th
and 5 th instar feeding larvae were incubated in 10 µg/ml Alexafluor 488 conjugated wheat germ
agglutinin (AWGA) (Invitrogen, Carlsbad, CA, USA) in phosphate buffered saline (PBS) (137
mM NaCl, 2.7 mM KCl, 10.1 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 ) for 30 min and then stored in
PBS at 4°C in the dark until examined by confocal microscopy. Confocal microscopy was
conducted using a Zeiss LSM 710 confocal microscope fitted with a 20X0.8 numerical aperature
objective lens. Zeiss ZENN 9.0 software was used to control the microscope. The Alexafluor 488
was excited using either an Argon LGN3001 488 nm laser with the pinhole set at 1 Airy unit or a
Coherent Chameleon laser tuned to 720 nm with the pinhole wide open. In either case, emissions
from 494-645 nm were collected, with a pixel dwell of 6.30 µs and line averaging set to 2. Voxel
size was between 0.4x0.4x3.0 µm and 0.5x0.5x3.0 µm (XYZ). Photomultiplier tube gain and
laser power were increased to maintain image brightness as imaging depth increased. Confocal
images were resolved using Autoquant X version 2.2.0 (Autodeblur Gold CF package) using the
default settings for three dimensional decovolution. Adjacent Z-stacks were then stitched using
XUV tools (http://www.xuvtools.org) (Emmenlauer et al., 2009) and visualized using Imaris
7.1.1 software (http://www.bitplane.com/go/products/imaris). Full resolution TIFF files were
captured showing the XZ plane to get a cross-section of the intact PM.
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