Biology Reference
In-Depth Information
stick tape is available in two types: ''permanent'' and a less aggressive
''removable'' (Scotch 667 from 3M). The less aggressive tape holds car-
riers without having to fight to remove them for loading in the freezing
holder, just prior to freezing. The specimen carrier can be filled with
bacteria and 1-hexadecene.
ii. HPF is preformed in a BAL-TEC HPM 010. Readers unfamiliar with HPF
and general issues related to successful specimen preparation should consult
McDonald (1999)
, including filling of specimen carriers, removal of the
specimen holder, and separation of specimen carriers.
iii. The use of a brass bottom and aluminum top (or vice versa) allows for quick
identification of the bottom carrier, which is transferred, while under LN
2
,to
a polypropylene freeze substitution vial filled with frozen 1% osmium tetrox-
ide and 0.1% uranyl acetate in acetone. The vial is capped and transferred to
an aluminum block also cooled in liquid nitrogen.
iv. The empty holes in the aluminum block are filled with liquid nitrogen and the
block is wrapped in aluminum foil and packed with crushed dry ice in a
Styrofoam box taped to a rotary shaker. The box is shaken at 100 rpm for 3 to
4daysat
80
C.
v. Shake vials on a microtiter plate shaker at 100 rpm for 2 to 3 days at
20
C.
vi. Warm to 4
C overnight, transfer to room temperature, and rinse with three to
four changes of dry acetone. Agarose blocks can usually be identified by the
presence of the embryos.
c. Epoxy Infiltration and Polymerization for Agarose-Embedded Embryos
i. Transfer freeze substituted specimens into 20-mL scintillation vials (wash
and oven dry) containing 30% Epon in acetone (EM grade) rotating on a
rotary mixer for 4 h to overnight at room temperature.
ii. 50% and 75% Epon in acetone for 2 h each at room temperature.
iii. Three changes of 100% Epon for 1 h each at 50-60
C.
iv. Transfer the resin-infiltrated agarose pad to a Rain-X or Teflon coated slide
with some fresh resin. Place two thicknesses of Parafilm on both ends of the
slide as a spacer and place a second coated (Rain-X) slide over the Epon-
infiltrated agarose.
v. Polymerize on a flat surface in a 60
C oven for 24 to 48 h.
vi. Remove the resin from between the slides, rough up one side by scraping a
razor blade across the surface, and mount/glue on a blank Epon block for
sectioning.
vii. One side of the now polymerized agarose pad containing embryos is
roughed up to remove residual Rain-X or Teflon release agent and to
increase the surface area for adhesion to an Epoxy blank. The small sample
piece can be attached to the blank using glue or additional Epoxy resin
followed by polymerization in a 60
C oven. This orients the embryos
parallel with the cutting plane to obtain a similar orientation in the TEM
as the view already obtained by LSCM.
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