The presence of Ki67-positive tumor cells and CD31-positive vessels in rat stroma tissue was analyzed using a fluorescence laser scanning system (TCS-SP2 scanning system and DM IRB inverted microscope, Leica®, Solms, Germany). Ki67 and CD31 staining were performed with specific antibodies as described above. Dye-coupled Alexa antibodies (Alexa-488 for Ki67 in green and Alexa-647 for CD31 in red; Molecular Probes, Eugene, USA) were used as secondary antibodies. Subsequently, Hoechst 33342 was used for labeling of nuclear DNA (Sigma®, Taufkirchen, Germany). Leica Confocal Software Lite (Leica®, Solms, Germany) was used for evaluation according to the manufacturer’s instructions.
Tcs sp2 scanning system
Manufactured by Leica
Sourced in Germany
The Leica TCS-SP2 is a scanning system designed for confocal microscopy. It provides high-resolution imaging capabilities to support various research and analysis applications.
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2 protocols using tcs sp2 scanning system
1
Quantifying Tumor and Vascular Markers
2
Immunofluorescent Localization of EpCAM
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Cells were seeded on object slides and fixed using paraformaldehyde and methanol as previously described (Brock et al, 1999 (link)). Blocking was performed at room temperature for 30 min using normal goat serum. Primary antibodies were incubated overnight at 4°C, while Alexa-labeled secondary antibodies (Molecular Probes, Carlsbad, CA, USA) were incubated for 1 h at room temperature. Antibodies used were anti-EpCAM (1 : 1000, clone C10, Santa Cruz Biotechnology=anti-EpEX) and anti-EpCAM (1 : 500, polyclonal guinea pig, peptide specific laboratories=anti-EpICD). Pictures were analysed in a fluorescence laser scanning system (TCS-SP2 scanning system and DM IRB inverted microscope, Leica, Wetzlar, Germany).
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