High-Pressure Freezing and Low-Temperature Processing of Plant Tissue Samples for Electron Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 147

Fig. 1 ( a ) Dissection of maize kernels and loading of basal parts of the endosperm specimens into HPF planch-
ettes. Steps in dissecting and loading are explained in the text. SE starchy endosperm, BETL basal endosperm
transfer cell layer. ( b ) Semi-thin section (500 nm) of a BETL and the nucellar placento-chalazal cell (NC) zone
of a maize kernel processed by HPF/FS. NC and the underlying integument were damaged during dissection,
as outlined by the dashed box . ( c ) TEM micrograph of a basal endosperm transfer cell in which a secretory
protein was immunolabeled (gold particles—15 nm). A Golgi/trans-Golgi network complex is marked with an
arrow . V vacuole, WIG cell wall ingrowth. Scale bar: 1 μ m

3. Add 20 % dextran dissolved in the BY-2 culture medium to the
cell slurry and gently mix ( see Note 4 ).
4. Pipette out the excess dextran solution, and transfer the result-
ing cell slurry to an A-type HPF planchette. Cover with the
fl at side of a B-type planchette.
5. Freeze and recover samples as described in Subheading 3.2 .

3.4 High-Pressure
Freezing (Developing
Maize Kernel)

1. Harvest maize ears at the particular stages of interest and bring
them to the lab quickly. Have the HPF machine ready before
harvesting ears.
2. Remove husks and pluck individual kernels from the ear using
a knife tip or other tool, without damaging the kernels ( see
Note 5 ).
3. Cut a kernel into approximately 2-3 mm slices (Fig. 1a -1).
Using a 2 mm biopsy punch, extract the endosperm tissues
(Fig. 1a -2). Slice the tissue thinly enough for fi tting into a
B-type planchette (Fig. 1a -3).
4. Transfer the tissue to a B-type planchette and cover with
another B-type planchette (Fig. 1a -4).
5. Freeze and recover samples as described in Subheading 3.2 .