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SleepyHollow02
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Untersuchte Arbeit:
Seite: 48, Zeilen: 10-24
Quelle: Gu et al 2009
Seite(n): 0, Zeilen: 0
3.2.10. TUNEL assay

The colonic cancer tissue was cut to 8 μm frozen sections and subsequently fixed in 4 % paraformaldehyde for 30 min at room temperature. After rinsing with PBS the samples were permeabilized in a solution of 0.1 % Triton X-100 in sodium citrate for 2 min. Samples, washed with PBS, were then incubated in the TUNEL reaction mix for 1 h at 37oC, according to the manufacturer’s instructions (Roche, Germany). Nuclei were stained with DRAQ5™ (Biostatus Limited). Sections were analyzed with a confocal laser scanning microscope (Carl Zeiss).

3.2.11. APOPercentage apoptosis assay

Caco2 cells were cultured in 96-well plates for the APOPercentage apoptosis assay (Biocolor Ltd., Belfast, Ireland). In the presence or absence of 10-6 M anastrozole (Sigma), they were stimulated or not with 10-6 M TAC for 24 hours in serum containing medium. Untreated cells cultured in serum free medium were used as positive control for the apoptotic response.

APOPercentage apoptosis assay Caco2 cells (in RPMI 1640, supplemented with 25 mM HEPES, 2 mM L-Glutamine and 10% FBS) were cultured in 96-well plates for the APOPercentage apoptosis assay (Biocolor Ltd., Belfast, Ireland). In the presence or absence of 10-7 M flutamide, cytochalasin B (Sigma) and DEVD-fmk, they were stimulated or not for 24 h in serumcontaining medium with 10-7 M of the following steroids: testosterone-HSA, dihydrotestosterone (DHT) and estradiol (E2). Untreated cells cultured in serum-free medium were used as positive control for apoptosis.

TUNEL assay The colonic cancer tissue was cut to 8 μm frozen sections from mouse colon tumors and subsequently fixed in 4% paraformaldehyde for 30 min at room temperature. After rinsing with PBS the samples were permeabilized in a solution of 0.1% Triton X-100 in sodium citrate for 2 min. Samples were washed with PBS and incubated in the TUNEL reaction mix for 1 h at 37°C, according to the manufacturer's instructions (Roche, Germany). Nuclei were stained with DRAQ5™ (Biostatus Limited). Sections were analysed by confocal microscopy.

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