Solid tissue sections can be analyzed for tumor-specific antigens or other fluorescent markers in order to determine the percentage and location of positive cells. By selectively gating positive cells or assigning a color to the cells of interest, the antigen-bearing cells can be mapped to visualize tissue architecture, or relocated by CompuSort for image capture.  

 tumor cells expressing 
melanoma antigen
In order to compare the DNA content of tumor cells with normal granulocytes, cells are first selected by a region of uniform cell size. The cell map is used to identify two tumor component regions, labeled blue and green. Colorized histograms can be overlaid to compare the DNA content of the three populations.


Cells expressing the melanoma antigen, HMB-45 in the cytoplasm are colored green and are localized to a single tumor region in the tissue map.

 

Infiltrating granulocytes are identified by their relatively small nuclear area and high PI Max Pixel. They are colored red and are localized in the region surrounding the tumor in the tissue map.

 

 

Staining Procedure
A 5 micron thick section of formalin-fixed dermal tissue was deparaffinized by placing the slides in a series of 6 baths of xylene, EtOh, (100,70, 50, 20%) and PBS for 5 minutes each. The slide was reacted for 30 min. with the anti-human melanoma protein antibody, HMB-45 (Dako Corp., Carpenteria, CA) diluted 1:20 in PBS with 1% BSA, rinsed with PBS/BSA 5 times, then developed with FITC-conjugated GAM (Dako Corp., Carpenteria, CA). The FITC signal was amplified with anti FITC antibodies (Dak 4, Dako Co., Carpenteria, CA) followed by GAM-FITC according to the manufacturers directions. The slide was counterstained by placing 50 µl propidium iodide with RNAse (PI, 5-50 µg/ ml depending upon the strength of FITC staining for a particular specimen) on the slide for 30 min. The slide was coverslipped using 40 µl of a mixture of PI in 50% glycerin/PBS at a PI concentration 5-50 µg/ml and stored in the dark prior to scanning.
LSC
Analysis
The tumor-rich area of the slide was selected and scanned using the 20X objective and the argon laser operating at 5 mW. Individual cells were segmented and contoured using PI fluorescence and a 100 pixel minimum area threshold. Perimeter contouring for the HMB-45FITC signal was selected in the area from 4-20 pixels outside the nuclear contour.

Copyright © 1998. All rights reserved. CompuCyte Corporation