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be different from cells at the tumor periphery. To identify novel
biomarkers by quantitative proteome analysis, the study design is
of high importance:
1.
It is mandatory to select a relevant tumor subtype and control
tissue. In the presented study, we analyzed SCC tissue.
Because SCC typically originates from bronchial epithelium
cells which are transformed in a multistep process including
metaplasia and dysplasia (
1
), we choose bronchial epithelium
as control tissue.
2.
Another important point is the isolation of relevant cells (
2
).
For this purpose, we used manual microdissection of tissue
slices. We collected cells from bronchial epithelium as control
tissue and separated tumor cells from surrounding stroma,
blood vessels, and nonmalignant tissue. Here, cell areas of
grade 2 (moderately differentiated) as well as grade 3 (poorly
differentiated) were included.
The selection of relevant cells for the analysis reduces sample
heterogeneity because the resulting protein sample is not “diluted”
by proteins from irrelevant cells, leading to an increased experi-
mental sensitivity.
Because microdissection is a time-consuming method, only
small amount of cells can be collected in reasonable time. Therefore,
DIGE saturation labeling is the method of choice analyzing small
cell numbers (1-5
μ
g protein) (
3, 4
).
2. Materials
2.1. Microdissection
and Sample
Preparation
1.
Microscope: BH2 (Olympus, Wetzlar, Germany).
2.
Cryostat: Shandon Cryotome SME (Thermo Scientifi c,
Dreieich, Germany).
3.
Ultrasonic bath.
4.
Hand homogenisator.
5.
Injection needle: 0.65 × 25 mm.
6.
Lysis buffer: 2 M thiourea, 7 M urea, 4% CHAPS (3-[(3-chol-
amidopropyl)dimethylammonio]-1-propanesulfonate), 30 mM
Tris-HCl, pH 8.0.
7.
Ethanol, 96%.
8.
25% (w/v) hematoxylin, according to Mayer (Merck).
9.
1.7% (w/v) eosin, diluted in 96% ethanol.
10. Bradford Protein Assay (Bio-Rad).
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