Ultrastructural Analysis of Bacteria–Host Cell Interactions - Bacterial Pathogenesis: Methods and Protocols - page 169

2.2. Conventional SEM
1. Same as items 1-7 and 12-14 in Subheading 2.1 (TEM).
2. Silicon chip specimen mounts (Ted Pella, Inc).
3. 24-Well tissue culture plates.
4. SEM sample stubs compatible with the microscope (Ted Pella, Inc).
5. Chromium, iridium, or other appropriate sputtering target (Refining Systems, Inc.,
Las Vegas, NV).
6. Carbon sputtering target (South Bay Technology, San Clemente, CA).
2.3. Immune Labeling with Quantum Dots
1. Phosphate-buffered saline (PBS).
2. Immunoglobulin-free bovine serum albumin (BSA).
3. Primary antibodies or anti-serum, for example, rabbit anti-serum.
4. Secondary antibody conjugate, for example, goat anti-rabbit 655 nm quantum
dots (Invitrogen, Inc., Carlsbad, CA).
5. Parafilm® or similar laboratory film.
6. Anti-capillary tweezers (Ted Pella, Inc).
3. Methods
3.1. Conventional TEM
This section describes steps for fixating and contrasting, dehydrating,
embedding, sectioning, and mounting samples for TEM. Many variations on
these steps have been described depending on types of samples used, experi-
mental goals, and personal preference (1,2,3,4) . This set of methods was used
successfully to demonstrate stages of infection of primary human neutrophils
with clinical isolates of group A Streptococcus , and methicillin-resistant Staphy-
lococcus aureus (MRSA), and lymphocytes by Borrelia burgdorferi . The
samples of neutrophils were allowed to adhere to 13-mm Thermanox® cover
slips in the wells of a 24-well tissue-culture plate prior to exposure to bacteria.
In contrast, 10 5 B cells and 10 6 spirochetes were co-incubated within liquid
growth medium suspensions and then processed for TEM as pellets. In these
examples, the time periods for each step are relatively long and reflect allowance
for slow diffusion of reagents through Gram-positive cell walls, and pellets
of cells and spirochetes, respectively. In all processing steps, care must be
taken to avoid unintentional drying of samples ( see Note 1 ). When necessary,
centrifugation and resuspension should be conducted as gently as possible to
minimize mechanical damage ( see Note 2 ). Furthermore, TEM grids must be
kept clean and handled very carefully, grasping only at the edges with very
fine tweezers.