This article details the macrodissection technique for isolating cancer tissues from specimens using hematoxylin and eosin (H&E) staining. The process involves careful identification and scraping of tumor areas to prepare samples for downstream analysis.
To begin macrodissection, take a glass slide carrying a tissue specimen. Additionally, take a reference slide carrying the same tissue specimen and stain it using hematoxylin and eosin for H&E stain.
In normal cells, hematoxylin imparts a blue stain to nucleic acids, while the eosin counterstain shows the cellular proteins pink. The deep blue coloration of the nucleus helps in differentiating cancer cells from normal cells.
Overlay the H&E stained slide with the unstained slide to align the tissue specimens. Locate the portion containing tumor cells on the H&E stained slide. Mark the corresponding area on the unstained slide.
Scrape off the desired tissue from the unstained slide. Using a wet pipette tip, collect the scraped tissue to ensure an easy transfer of the tissue specimen. Transfer the tissue into a tube containing a lysis buffer. Components of the lysis buffer digest the tissue, releasing the constituents of the cell.
Treat the lysate with a suitable protease and incubate at a high temperature. The protease cleaves the contaminating DNases, thereby protecting the DNA from degradation. Store the DNA containing tissue lysate for downstream analysis.
Identify cancer tissues of the tumor area most suitable for macrodissection according to the appropriate H&E stained section. Then, macrodissect the cancer tissue based on the marked section. Take a clean razor blade and gently scrape the cancer tissue off of the slide, trying to keep it in one piece. Use the pipette tip to transfer the scraped tissue into the lysis buffer vial.
Repeat this process with the rest of the slides. After all the tissue is in the tube, use the tip to make sure the tissue is fully submerged and is not stuck to the wall. Add 20 microliters of a subtilisin-related serine protease to the vial and gently flick to mix. Place the vial in a 55 degree Celsius heat block for at least 4 hours or overnight, making sure to slightly vortex after 2 hours.
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