Resin Embedding, Sectioning, and Immunocytochemical Analyses of Plant Cell Walls in Hard Tissues - Plant Cell Morphogenesis: Methods and Protocols - page 46

Fig. 2 Sample preparation and positioning for sectioning with an ultramicrotome.
( a ) Align block parallel to the cutting direction of the knife ( k ). For acrylic resin,
such as LR White, the water level ( blue ) should form a concave meniscus in the
boat. ( b ) Align block face parallel to the knife. ( c ) Ensure upper and lower sides
of the block are parallel with the knife. ( s ) = sample in block (color fi gure online)

2. Using a fresh stainless steel razor blade, trim as much resin
from the block as possible to produce a trapezium leaving your
sample in the middle of the block face with the widest edge of
the trapezium at the bottom of the block. The upper and
lower edges of the block should be parallel with the knife
edge. The procedure is outlined in Fig. 2 .
3. For light microscopy, cut sections to a thickness of 0.5-2 μ m

onto water.
4. Transfer sections to a drop of water on Vectabond-coated
Multitest slides and dry sections onto the slide using a slide
drying hot plate. It may be useful to analyze some of the sec-
tions under the light microscope by staining with aqueous 1 %
(w/v) Toluidine Blue O containing 2 % (w/v) Borax (fi ltered
before use) to determine section quality and that the features
of interest are present.
5. For electron microscopy, cut ultrathin sections to a thickness
of ~80 nm when they appear silvery gold in color and collect
sections on nickel grids.
For troubleshooting issues with sectioning see Note 6 .

These procedures are for the indirect immunofl uorescence label-
ling of sections of plant material ( see Note 7 ). Always include a
negative control (omission of the primary MAB) to assess the
extent of cell wall autofl uorescence present in the sample. Here we
focus on immunofl uorescence procedures, but there are very effec-
tive alternatives such as immunogold with silver enhancement for
light microscopy [ 7 ]:

3.4 Immunolabelling
of Sections
Using MABs

1. Use the PAP pen to isolate individual wells on the Multitest
slide.
2. Block nonspecifi c binding sites by incubation with PBS/MP
for at least 30 min (a 30 μ l volume is suffi cient to ensure cover-

age of the sample).