Biology Reference
In-Depth Information
the resin, to separate them (
Fig. 20.3
E). This must be performed with extreme care, as
too much leverage by the razor will shatter the glass. This shattering usually renders
the sample unusable, as removing all the glass fragments from the resin is very dif-
ficult without damaging the sample, and any microscopic shards of glass remaining
will damage the diamond knife during sectioning. Dipping the resin and dish into
liquid nitrogen for 1-2 s can help separate them, as the difference in thermal expan-
sion between resin and plastic will eventually detach them. Other protocols use 40%
hydrofluoric acid to dissolve the glass, bypassing this tricky step (
Polishchuk,
Polishchuk, & Luini, 2012
).
Once detached, excess resin can be trimmed away until only the capsule remains
(
Fig. 20.3
F and G). The coordinates imprinted on the underside of the capsule can be
observed using a tissue dissection microscope; draw around the coordinate contain-
ing your cell of interest using a thin marker pen, the LM images serving as reference
(
Fig. 20.3
H). Using a microtome chuck and bench-top vice to firmly hold the resin
block in place, remove excess resin around the coordinate using a junior hacksaw
(
Fig. 20.3
I), making sure that it never scratches the coordinate surface. This risk
can be minimized by trimming away the resin using razor blades (
Fig. 20.3
J).
The remainder of the resin trimming and sectioning is performed using an ultra-
microtome with glass knives (
Fig. 20.3
K) and a diamond knife (
Fig. 20.3
O), respec-
tively. It is possible to make out the cell of interest and the etched coordinate using
microtome binoculars (
Fig. 20.3
M and N). We routinely trim a square block face, up
to
50
m
m from the cell edge (
Fig. 20.3
L), but a wider space can be left according to
one's experience. The larger the block face created during sectioning, the more dif-
ficult it will be to locate the cell in the sections under the EM. A square block face is
optimal, as this helps acquire serial sections during sectioning. Sections 80 nm in
thickness are taken using the diamond knife and collected using the copper grids
coated with formvar (
Fig. 20.3
P and Q). To handle the grids, high-precision tweezers
should be used at all times, carefully gripping the grid by its outer edge only, so as to
not tear or damage the formvar or sample sections.
To attain optimal contrast under the microscope, we post-stain the sections by
placing each grid section-side-down onto a drop of 5% uranyl acetate in 50% ethanol
for 7-8 min, gently rinse in distilled water for 1 min, and place face-down on a drip
of Reynold's lead citrate solution for 7-8 min. The grid is then rinsed in water again
for 1 min. Both solutions should be centrifuged in 1.5-ml Eppendorf tubes at
and the dish base (D). Following the separation of resin and dish (E), excess resin was
removed using pliers (F) until just the capsule remained (G). The cell of interest was marked
(H) with the aid of LM images (M) and resin coordinates (N). Unwanted resin was removed
using a junior hacksaw (I) and a razor (J). Resin was trimmed using a microtome and a
glass knife (K) until a neat block was generated at the top of a pyramid (L). Blocks were
sectioned using a diamond knife (O) and ribbons collected using 100 mesh copper grids
(P and Q), coated with formvar.
