Biology Reference
In-Depth Information
6. Use whiteout solution to place marks close to the samples for
defi nition of sample position in the fl exImaging software
before starting the measurement run (
see
Subheading
5.4
).
7. Capture an optical image with a slide scanner or stereomicroscope
(e.g., Leica MZ6, Wetzlar, Germany) connected to a digital
camera (AxioCam ICc1, Zeiss, Jena, Germany) or a similar
image acquisition system (
see
Note 1
).
5.2 Spatially
Resolved Tryptic
Digest (Optional)
1. Reconstitute one vial (20
g) of trypsin (Sequencing Grade
Modifi ed Trypsin, Promega, WI, USA) using 180
μ
μ
l of ammo-
nium bicarbonate (20 mM) and 20
μ
l acetonitrile. Avoid
foaming.
2. Carefully adjust the global spray power offset of the ImagePrep.
Water or ammonium bicarbonate solution (20 mM) can be
used for training purposes. Using a spray power of 38 % with
no modulation, the offset should be adjusted until a faint but
continuous spray for 20-40 s can be achieved using a single
200
l droplet must be
directly loaded into the fl ow pipe of the image prep, and not in
the matrix reservoir. Detailed information is available from the
manufacturer.
3. Load 200
μ
l droplet. To achieve this, the 200
μ
l of trypsin solution as described under
Subheading
2
, and place the sample in the ImagePrep, posi-
tioning it on the central elevated platform. The scattered light
sensor should not be covered by tissue. Execute the trypsin
deposition protocol (
see
Note 11
). Thoroughly clean the
ImagePrep using the “Clean” option of the instrument with
pure methanol.
4. After trypsin deposition, carefully remove the slide from the
ImagePrep and place it in a suffi ciently large container fi lled
with several wet paper towels. Humidity should be high
enough to ensure effi cient digestion, but there should be no
water condensation on the ITO slide. Incubate the container
with the sample at 37 °C for 90 min.
5. After incubation, remove the slide from the container, return it
into the ImagePrep and follow the standard procedure for
depositing matrix on the slide.
μ
The following procedures have been optimized for the automated
deposition of matrix onto cryo-sections using the ImagePrep
device (Bruker Daltonics GmbH, Bremen;
see
Note 2
).
5.3 Matrix
Deposition for the
Analysis of Tryptic
Peptides
1. Fill the matrix vial with pure methanol and set the global spray
power adjustment for the ImagePrep as described in the manual.
2. Insert the slide with digested sample into the ImagePrep
instrument and adjust slide position on the small elevation
in the way that the light sensor isn't covered by a section.
