Ultrastructural Analysis of Bacteria–Host Cell Interactions - Bacterial Pathogenesis: Methods and Protocols - page 168

for electron microscopy without further manipulation of the label. This was
recently described for simultaneous detection of several quantum dot probes
by fluorescence and TEM of sections by the wavelength of emission and size
class of particles, respectively (7) . To date, there are no reports of SEM studies
using quantum dots as immune labels. However, phosphorescent emissions
similar in wavelength to photoluminescent emissions have been detected by
SEM with a cathodoluminescence spectrometer (13) . Studies are underway
to determine whether cathodoluminescent properties of quantum dots will be
adaptable for immune labeling of biological material for SEM. In the meantime,
the CdSe core and ZnS coating of quantum dots provide for compositional
contrast using backscattered electron detection. Labeling and imaging methods
described below enable direct detection of the quantum dots by TEM and
low-voltage SEM.
2. Materials
2.1. Conventional TEM
1. Hank's-buffered salt solution (HBSS) (Cambrex BioSciences, Inc., Walkersville,
MD).
2. 25% aqueous glutaraldehyde, EM grade, in nitrogen-filled ampules (Ted Pella,
Inc., Redding, CA).
3. 16% aqueous paraformaldehyde, EM grade, in nitrogen-filled ampules (Electron
Microscopy Sciences, Hatfield, PA).
4. Sodium cacodylate (dimethyl arsenate) (Ted Pella, Inc).
5. 4% aqueous osmium tetroxide, EM grade (Ted Pella, Inc).
6. Potassium ferrocyanide.
7. 70 and 100% (dehydrated) ethanol.
8. Spurr's embedding medium (Ted Pella, Inc).
9. Uranyl acetate (Ted Pella, Inc).
10. Lead citrate (Ted Pella, Inc).
11. 10 N sodium hydroxide.
12. Disposable serological pipets.
13. Disposable transfer pipets.
14. Powder-free nitrile or other appropriate gloves.
15. Thermanox® cover slips, 13 mm (Nalge Nunc International, Rochester, NY).
16. Embedding capsules or molds (Ted Pella, Inc).
17. Copper EM grids, 200-300 mesh (Ted Pella, Inc).
18. Grid box(es) (Ted Pella, Inc).
19. Laboratory film such as Parafilm®.
20. Syringe filters, 20 nm Anotop® (Whatman International, Ltd., Maidstone, UK).
21. Disposable Leur-Lock syringes, 10 mL.
22. Fine tweezers, such as anti-capillary tweezers.