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| 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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