Proliferation/apoptosis determination by tissue cytometry in gastrointestinal fresh frozen sections using triple labeling and automated scanning fluorescence microscopy

Jozsef Bocsi; Ferenc Sipos; Levente Ficsor; Viktor S. Varga; Zsolt Tulassay; Attila Tarnok; Béla Molnár

doi:10.1117/12.645823

21 February 2006 Proliferation/apoptosis determination by tissue cytometry in gastrointestinal fresh frozen sections using triple labeling and automated scanning fluorescence microscopy

Jozsef Bocsi, Ferenc Sipos, Levente Ficsor, Viktor S. Varga, Zsolt Tulassay, Attila Tarnok, Béla Molnár

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Proceedings Volume 6088, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IV; 60880I (2006) https://doi.org/10.1117/12.645823
Event: SPIE BiOS, 2006, San Jose, California, United States

Abstract

Proliferation/apoptosis balance is an important information in gastrointestinal ulcerative and malignant diseases. Immunohistochemical staining and visual counting is routine procedure. Recently we reported a new scanning fluorescence technique for automated motorized microscopes (SFM). Development of triple fluorescent labeling method for proliferating/apoptotic/resting cells and application of SFM for the automated analysis and counting on gastric biopsy specimen. Routine antral biopsy specimens by gastroscopy were fresh frozen and 5 micron sections were prepared. Proliferation was detected using a PCNA antibody, anti-mouse-biotin and streptavidin-Texas-Red labeling system. Apoptotic cells was labeled using the TUNEL reaction with FITC bound nucleotids. DAPI nuclear counter staining was applied. Labeled sections were scanned and digitized in the three fluorescent channels. SFM was modified to detect epithelial surface, glands in the biopsy specimen. Automated nuclei detection, PCNA and TUNEL detection was performed, ratio was calculated. In parallel standard biopsies were labeled with PCNA and AEC. TUNEL reaction was performed. Up to 1000 epithelial cells were manually counted. The mean PCNA labeling in healthy samples were 45,3±12,4%, that significantly increased to 56.4±8.7% in H. Pylori positive cases. Positive TUNEL reaction was found in 2,9±1,1% in H. pylori negative cases, while in the H. Pylori positive cases the apoptotic ratio was significantly increased (14.1±3.2%, p<0.05). Significant correlation in apoptosis/proliferation ratio between the SFM and routine methods could be observed (p<0,05). SFM procedure proved to be more time efficient both in labeling, both in detection procedures. Triple fluorescent labeling and automated fluorescence microscopy is an applicable tool for the proliferation, apoptosis determination in fresh frozen samples.

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Jozsef Bocsi, Ferenc Sipos, Levente Ficsor, Viktor S. Varga, Zsolt Tulassay, Attila Tarnok, and Béla Molnár "Proliferation/apoptosis determination by tissue cytometry in gastrointestinal fresh frozen sections using triple labeling and automated scanning fluorescence microscopy", Proc. SPIE 6088, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IV, 60880I (21 February 2006); https://doi.org/10.1117/12.645823

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