Biology Reference
In-Depth Information
The image analysis provides a list of
m/z
signals relevant in the
context of each specifi c analysis. At this point, additional experiments
are required to elucidate the chemical structure of the compounds.
These include accurate mass measurements, MS/MS analysis, on-
tissue digestion, or extraction and analysis of laser-dissected areas
containing the unknown compound of interest among other
approaches. The choice of an appropriate strategy will depend on the
specifi c experimental context.
5
Methods for MALDI-MSI of Proteins and Peptides
1. Clean the sectioning blade or put a new one into the cryostat
and adjust the cutting temperature. For most samples, the
temperature is set to −20 °C. If the non-embedded part of the
sample tends to be still fl exible touching the blade during sec-
tioning process, lower temperatures should be used. Otherwise,
for highly fragile samples test higher temperatures (i.e.,
−10 °C) (
see
Note 3
).
2. Fresh plant material has to be frozen in liquid nitrogen.
Transfer the frozen sample into the cryostat chamber cooled to
the desired temperature (−20 °C) and fi x it onto the sample
plate via either a droplet of water (ice) or of OCT medium (
see
Note 4
). If necessary, precut the plant material with a razor
blade to a suitable length before fi xation. Select a proper orien-
tation for longitudinal or cross sections.
3. Start the sectioning process by moving the cutting block with
the sample plate. To trim the tissue sample a large section
thickness can be selected until the desired morphological parts
are visible. Then change the setting to the desired thickness.
Cut 20-30
5.1 Sample
Preparation, Cryo-
sectioning, and Slide
Preparation
m for tobacco
roots (
see
Note 5
). Transfer the sections immediately onto
indium tin oxide (ITO) coated glass slides and fi x the sections
by thaw-mounting. By touching the opposite glass side with a
fi ngertip for ca 20-30 s the ice in the section is melted and
evaporated.
4. Transfer the ITO slides with the sections into a desiccator for
0.5-1.0 h until they are completely dried. The time until com-
plete dryness varies and is longest for the high water content
tissues (roots). Alternatively, for low water containing samples,
the drying step can be skipped.
5. After drying, samples must be subjected to a washing proce-
dure (
see
Note 10
). Immerse the slides in 70 % ethanol (two
times for 2 min each) and absolute ethanol (once for 2 min).
After washing, remove excess ethanol from the slides carefully
with a paper towel and return the slides to the desiccator for
another 0.5-1 h, until they have completely dried.
μ
m sections for seeds and 35-45
μ
