Sample Preparation
Nuclear Extraction. Three 50 micrometer sections are cut from the tissue block for nuclear extraction. A 5 micron section is cut from the block before and after the thick sections, stained with H & E, and examined to confirm the presence of representative tumor cells. Tissue sections are deparaffinized and rehydrated in xylene and a series of decreasing ethanol to water using a tissue processor. The tissue was incubated for 45 minutes at 37° C in 1 ml of 0.5% pepsin (pH = 1.5) in 1.1% sodium chloride and 3.5% polyethelene glycol with vortexing every 10 minutes. After pepsin digestion, samples are washed and resuspended to 1 x 106 /ml in phosphate buffered saline (PBS). A single drop of the cell suspension is deposited on a Poly-L-lysine coated glass slide, and incubated at room temperature in a humidity chamber for 15 minutes. A single drop of cold 95% ethanol is dropped on the cell suspension to quickly disperse the PBS. After the slide has dried, a drop of a 50 µg/ml solution of propidium iodide in PBS containing 200 µg/ml RNAse is added. After a 10 minute incubation, the slide is covered and analyzed immediately.
Tissue Section. Five-micrometer thin sections are cut from paraffin block and placed on 3-aminopropyltriethoxysilane (APES) coated slides and allowed to dry. The sections are then deparaffinized and rehydrated in xylene and a series of decreasing ethanol to water. A drop of a 50 µg/ml solution of propidium iodide in PBS containing 200 µg/ml RNAse is added to the slide. After a 10 minute incubation, the slide is covered and analyzed immediately.
LSC Analysis
Nuclear Extraction. After establishing the scan area, the slide is analyzed using a 40X objective and 5 mW of Argon laser power. A minimum of 15,000 cells (or the entire cell preparation) is examined. A cell gallery is created by relocation of cells from each of the major peaks in the histogram of integrated red fluorescence. The morphologic composition of relocated cells is examined for purposes of quality assurance.
Tissue Section. After establishing a scan area, the slide is analyzed using a 40X objective and 5 mW of Argon laser power. Due to the close proximity of nuclei in section, threshold parameters alone cannot control segmentation and detection of a single nuclei. Unlike image analysis, there are no provisions for “cutting” nuclear aggregates to define single nuclei for analysis by LSC. Aggregates of sectioned nuclei interfere with histogram interpretation. Therefore, it is necessary to establish a gating routine to exclude these nuclear aggregates. This is best demonstrated with rat liver, which contains uniform populations of diploid and tetraploid nuclei (no cycling cells).
 .....The DNA histogram prepared by LSC analysis of an imprint from rat liver shows two distinct peaks corresponding to DNA diploid and DNA tetraploid cells (Figure 3A). The plot of red sensor peak verses area (Figure 3B) shows a gate designed to eliminate aggregates of nuclei. In this touch imprint, there are few aggregates, and the threshold parameters alone adequately control segmentation of single nuclei.
.....The corresponding DNA histogram generated from rat liver tissue section shows three distinct broadened peaks corresponding to diploid, sectioned tetraploid, and nearly whole tetraploid nuclei (Figure 3C). The middle peak represents the so-called shadow peak which is commonly observed in tissue section image analysis. Note that a number of aggregates have been gated out of the DNA histogram through the plot of red sensor peak verses area (Figure 3D). Relocation can be used to confirm that the nuclei, which make up each of the peaks in the DNA histogram are single in nature.
Procedure and data from Dr. James B. Hendricks, Children’s Hospital Research Institute, New Orleans, LA |
