Anti ck20 antibody

Formalin-fixed, paraffin-embedded sections derived from the xenografts were stained with anti-human cytokeratin 20 (CK20) mouse monoclonal antibody (Clone: Ks20.8, dilution 1∶25, Dako, Glostrup, Denmark), anti-human cytokeratin 7 (CK7) rabbit monoclonal antibody (Clone: SP52, concentration; 0.536 µg/ml, Roche) or anti-human caudal type homeobox 2 (CDX2) mouse monoclonal antibody (Clone: AMT28, dilution 1∶500, Leica Biosystems, Wetzlar, Germany) by the avidin-biotin immunoperoxidase method. Microwave antigen retrieval was performed. For immunocytochemistry, the cultured cells, which were fixed with 4% paraformaldehyde, were stained with anti-CK20 antibody (Clone: Ks20.8, dilution 1∶100) or anti-CDX2 antibody (Clone: CDX2-88, prediluted, Abcam), and counterstained with Hoechst33342 (Life Technologies) to identify all nuclei.

TUNEL assays were performed using an In Situ Cell Death Detection Kit, POD (Roche Applied Science, Mannheim, Germany) [22] (link)–[25] (link). Additionally, colon sections were stained with a rabbit polyclonal anti-mouse cytokeratin 20 (CK20) antibody (Abcam, Cambridge, England) to highlight the epithelial lining of the colonic mucosa. DSS-injured colons on day 15 were labeled with anti-fluorescein antibodies (brown-stained) (TUNEL POD; Roche Applied Science), or fluorescein-conjugated TUNEL reaction mixture (green-stained) (Roche Applied Science) and a rabbit polyclonal anti-CK20 antibody (1∶200; Abcam). For the latter, the staining was visualized with donkey anti-rabbit IgG antibodies conjugated with Alexa Fluor Dyes (red-stained) (Invitrogen) by confocal laser scanning microscopy (LSM5 Pascal Exciter) (original magnification: ×400) [22] (link), [24] (link). For a quantitative analysis, we counted the TUNEL⁺ lining epithelial cells (brown-stained) in 100 randomly selected crypts per section (original magnification: ×400) [22] (link)–[25] (link).