Immuno-Fluorescence SEM
Figure 2: U373 glioblastoma exosomes labeled with Qdot 565 bound to anti-EGFr (see Cell Handling & Labeling) (green) are bound to the membrane veil of a monocyte of the immune system. The electron image (gray-scale), from a mix of the upper and lower electron detectors, is overlaid with the CL image (green). (Inset) The cell from which the enlargement was taken.
while avoiding an increase in autofluorescence [17]. We use fresh, buffered, pH 7.4, room temperature, formaldehyde (2%) for 30–45 minutes [each cell type should be tested for optimal fixation techniques], followed by a buffer wash using slow pipetting. Te supernatant/monocyte samples were labeled with mouse anti-human EGFr (Invitrogen, Carlsbad, CA) and secondarily labeled with goat anti-mouse Qdot 565 (Invitrogen). Some monocytes were labeled, straight from the isolation procedure, with mouse anti-human CD14 (Invitrogen) and secondarily labeled with goat anti-mouse Qdot 605 (Invitrogen). In the case of these experiments, we used Qdots as secondary labels because the primary labels had been obtained for other experimental applications. Spatial resolution will, of course, be improved with the use of only primary labels. Te monocytes prepared for confocal imaging were handled just as those used for the exosome/monocyte cathodoluminescence except that CMFDA, a cytoplasmic dye (Invitrogen), was added to the monocytes before incubating with the tumor supernatant.
Sample Preparation Generally, SEM samples are sputter-coated with gold/
palladium (or similar metals) to avoid a charge build-up on the sample surface and subsequent saturation of the electron detector. However, like other workers [7, 18–20], we have noted almost complete loss of fluorescence with a 60-second gold/ palladium coating. We found that a 15-second sputter coating passes at least 50% of the maximum fluorescence. To reduce surface charging while reducing the extent of gold coating, we mount samples on aluminum dishes. Te dishes we use (
ScienceGear.com, P/N MWD-3500) fit into the wells of a 24-well culture plate and are approximately the same diameter as the aluminum studs commonly used on SEM stage mounts (Figure 3). Cell culturing, labeling, fixing, and drying can all be done in the aluminum dish. Aſter fixing and drying the cells, we affix the dish to the stud with a double-stick carbon spot, flange the edge of the dish with a small clipper, and fold down the edges to make a tight contact with the stud to ensure charge dispersal. Te ethanol wash prior to critical point drying [21] must also be kept to a minimum time to avoid membrane loss
2010 September •
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Figure 3: Aluminum dishes used for mounting, fixing, and coating cells (A). Dishes (arrow) fit into 24-well culture plates for cell growth incubation and are coated with poly-L-lysine or a similar cell growth binder to attach cells to the surface; tabs are convenient for lifting out dishes. (B) After cell processing, prior to the drying protocol, tabs and sides of dishes are trimmed and then attached to standard aluminum stage stubs (arrow) by double-sided adhesive carbon tape. (C) Flanges are cut into the side of the dish and bent under for maximum grounding contact with the stud.
and, thus, loss of surface markers. Usually the ethanol wash is performed in multiple steps of increasing ethanol concen- tration. We try to limit the step-up of ethanol to one or two minutes per step followed as quickly as possible by the critical point dryer.
Instrumentation A Hitachi S-4700 FE-SEM (Hitachi, Schaumburg, IL)
was used in the dual imaging mode, collecting secondary (SE) and backscattered (BS) electrons on the two built-in electron detectors and collecting photons on the Centaurus cathodo- luminescence detector (KE Developments Ltd, Cambridge, UK). Of particular importance is the use of a setting on the Hitachi S-4700 termed “analysis mode,” which changes the cross-over of the beam to a larger cross section, increasing the beam current about 10-fold, at the expense of some loss in image resolution. Te condenser lens notch should be set to as small a value as possible consistent with an acceptable SE resolution (Tis setting is the negative logarithm of the specimen beam current, thus yielding a higher current from a lower notch number. We use a setting of 4.) Under this mode we use an accelerating voltage of 30 kV and an emission current starting at 40 µA, either or both of which may be decreased if the photon output of the fluorophore is high enough. Accelerating
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