Ultrastructural Analysis of Bacteria–Host Cell Interactions - Bacterial Pathogenesis: Methods and Protocols - page 170

3.1.1. Fixation
1. Immerse samples in at least 10 volumes of Karnovsky's fixative containing 2.5%
glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH
7.2for2h( see Note 3 ).
2. Wash the samples twice for 30 min each time by replacing the fixative with
cacodylate buffer.
3. Post-fix the samples by replacing the wash buffer with a mixture containing 1%
osmium tetroxide and 0.8% potassium ferrocyanide in 0.1 M cacodylate for 2 h.
4. Wash the samples once for 30 min in cacodylate and twice in deionized or distilled
water.
3.1.2. In-Block Staining (Optional)
1. Stain the samples by immersion in 1% uranyl acetate in water for2hatroom
temperature.
2. Wash the samples twice for 30 min each in water.
3.1.3. Dehydration and Embedment
1. Dehydrate the samples by immersion for 1 h each in 70, 100, and 100% ethanol.
2. Prepare embedding resin according to the manufacturer's instructions and mix
thoroughly.
3. Infiltrate samples with embedding resin by immersing in 1/3 resin and 2/3 ethanol
for 4 h, 2/3 resin and 1/3 ethanol for 4 h, and 100% resin for 8-16 h twice.
4. After two cycles in 100% resin, transfer the samples to resin-filled embedding
capsules or other suitable molds designed for curing at elevated temperature.
Orient the samples for the optimal position and angle of sectioning. For centrifuged
material, this is typically not an issue due to the random orientation of cells
and bacteria within the pellets. For adherent cells on Thermanox® cover slips,
Aclar®, or silicon chips, orient the substrates such that the cells face toward the
resin at the preferred angle for sectioning, usually either parallel or perpendicular
to the cell layer. It is helpful to place a small paper label within the resin block
with identifying information written in pencil or printed in a small font (e.g.,
5 point font) for later reference.
5. Carefully place the samples into an oven for curing at 65 °C or as recommended
by the resin supplier ( see Note 4 ). Curing is temperature dependent. At 65 °C,
blocks are usually hardened within 24-48 h.
3.1.4. Block Face Preparation and Sectioning
1. Remove the cover slip or any other substrate from the block ( see Note 5 ). The
cells should remain in the block, and exposed on the surface.
2. Secure the block in a microtome chuck or small vice. Using single-edge razor
blades, carefully trim the block down to a suitable block face for sectioning. This
is typically a trapezoid ranging in size from about 0.2-1.0 mm on each side.