The changes that cancer cells undergo can be described as those of chaos: variability without order. Cancer cells are marked by differences in their size and shape, differences in the appearance of their nuclei, and abnormalities in their nuclear chromatin texture.

To identify neoplastic cells in a stained tissue section, look for differences in nuclear and/or cell size from one cell to another. Also look for irregularities in shape and size; normal tissue is regular and uniform, while malignant growth is usually jumbled and disorganized (Figure 1).

Figure 1. Normal colon vs. colon cancer. (Cresyl violet, no coverslip, Ambion protocol, ready for LCM harvesting)

Differences in the shape of cells and nuclei can be obvious, but may not be so due to the many different cell types present in a tissue section.

Cancer cells often have abnormal mitoses. Note the variation between nuclei in Figure 2.

Figure 2. Colon cancer. Note the disorderly nuclear pattern. Also note that the nuclei are larger than the normal above, are much more variable in size (circle), and that there is a mitotic figure (arrow). (Cresyl violet, no coverslip, Ambion protocol, ready for LCM harvesting)

Researchers accustomed to viewing routine H&E-stained material will note the different appearance of material prepared for LCM. This is not due to poor staining, but is an artifactual appearance created as the tissue is frozen and dehydrated. The lack of a coverslip over the tissue will drastically alter the appearance. Think of a piece of fine hardwood that has been cut and stained, but not varnished. You can see the grain, but not with the depth and luster that you would if it were thickly varnished. That is what a coverslip and mounting media do for a tissue section—give it the depth and luster.

To familiarize yourself with this appearance, we suggest using a serial section technique. Place two frozen-sections, one level then the next, on a single slide. Physically separate the two sections with a barrier pen. Stain one section with Cresyl Violet and cover with a coverslip. Stain the other section with Cresyl Violet or Acridine Orange and leave it uncovered. Use the section with a coverslip to orient yourself, then use the uncovered section to harvest your target by LCM.

There is an easy way to orient yourself from one section to another. Remove the ocular lens from your microscope and turn it around so that you are looking through it backwards; it will serve as a magnifying lens. Using this magnifying lens mark a dot on a very recognizable landmark on both sections. Use this mark as a reference point for navigation.

Acridine Orange stain is recommended when the tissue does not stain well with Cresyl Violet. Tissue that has been poorly handled, improperly frozen, or tissues that are inherently difficult to work with (e.g., lung, pancreas, liver) may be visualized better with Acridine Orange. One advantage of Acridine Orange is that it provides good contrast between the nuclei of the cells and the other cellular components. Since cancer cells are more likely to have big, jumbled and prominent nuclei, it provides a good outline of structures you may want to harvest with LCM.

Figure 3. Colon cancer. Image at left is Cresyl violet-stained, NOT coverslipped. You can also coverslip this section for even better morphology and use it as a guide for the Acridine Orange-stained section to the right that is without a coverslip, ready for LCM harvesting. (Cresyl violet, no coverslip, and Acridine stain, no coverslip, Ambion protocol, ready for LCM harvesting)

If you are having difficulties with staining or differentiation, try the serial section technique with stains that you are familiar with. Place one section on a slide, stain it with your favorite stain, and coverslip it. Next to it place a serial section stained with the dyes provided in Ambion’s LCM Staining Kit. Switch views from one to the other until you become familiar with the appearance of the LCM-stained material.

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