ffpe tumor tissue macrodissection: a technique to obtain specific tumorous tissue from unstained specimens

FFPE Tumor Tissue Macrodissection: A Technique to Obtain Specific Tumorous Tissue from Unstained Specimens

Identify cancer tissues of the tumor area most suitable for macrodissection according to the appropriate H&#38;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.

ffpe-tumor-tissue-macrodissection-technique-to-obtain-specific

Research

JoVE Encyclopedia of Experiments

Cancer Research

Gastrointestinal Cancer

Assays and techniques

EoE Sub Articles

eoe sub articles

1. Scraping the Slides 

<ol>
	<li>Prepare digestion/lysis buffer with 650 mL of diethylpyrocarbonate (DEPC)-treated water, 100 mL of ethylenediaminetetraacetic acid (EDTA), 50 ml of Tris hydrochloride (Tris-HCL) 2 M pH 8.8, and 200 mL of 10% sodium dodecyl sulfate (SDS) (see Table of Materials).</li>
	<li>Fill a 1.5 mL single-use polypropylene tube with 80 &#181;L of digestion/lysis buffer (see Table of Materials).</li>
	<li>Put a clean pipette tip into the buffer.</li>
	<li>Identify cancer tissues of the tumor area most suitable for macrodissection according to the appropriate H&#38;E-stained section.</li>
	<li>Macrodissect cancer tissue based on the marked H&#38;E-stained section.
	<ol>
		<li>Take a clean razor blade and gently scrape the cancer tissue off the slide, trying to keep it in one piece so that it is easier to work with.</li>
		<li>Take the pipette tip out of a single-use polypropylene tube containing buffer and use it to transfer the scrapped tissue into the buffer vial (see Table of Materials). 
		NOTE: The wet tip should attract the tissue, which makes the transfer easier.</li>
	</ol>
	</li>
	<li>Repeat step 1.5 with the rest of the slides.</li>
	<li>After all the tissue is in the single-use polypropylene tube, use the tip to make sure the tissue is fully submerged and is not stuck to the wall of the tube.</li>
	<li>Add 20 &#181;L of a subtilisin-related serine protease to the vial and gently flick to mix (see Table of Materials).</li>
	<li>Place the vial in a 55 &#176;C heat block for at least 4 h or overnight. Make sure to slightly vortex after 2 h.</li>
</ol>

<table><tbody><tr><td>10% Sodium dodecyl sulfate (SDS)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Quality Biological</td><td>351-032-721 EA</td><td>Step 2.1.</td></tr><tr><td>DEPC-treated water&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Quality Biological</td><td>351-068-131</td><td>Step 2.1.</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)&amp;nbsp;&amp;nbsp;</td><td>Corning</td><td>46-034-CL</td><td>Step 2.1.</td></tr><tr><td>Tris hydrochloride (Tris-HCL) 2 M pH 8.8&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Quality Biological</td><td>351-092-101</td><td>Step 2.1.</td></tr><tr><td>Proteinase K&amp;nbsp;</td><td>New England Biolabs</td><td>P8107S&amp;nbsp;</td><td>Step 2.8.</td></tr><tr><td>Single-use polypropylene (Eppendorf) tube</td><td>Eppendorf</td><td>24533495</td><td>Step 2.5.2.</td></tr></tbody></table>

Source: Sugimoto, K. et al. <a href="https://www.jove.com/video/61355" target="_blank">Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer.</a> J. Vis. Exp. (2020)
This video describes a method for the manual macrodissection of cancer tissue from unstained tissue specimens to obtain a specific cancer tissue. The tissue section can be processed further to obtain DNA for genome-wide studies on gastrointestinal cancers.

Source: Sugimoto, K. et al. Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer. J. Vis. Exp. (2020)
This video describes a method for the ...

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&#38;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&#38;E stained slide with the unstained slide to align the tissue specimens. Locate the portion containing tumor cells on the H&#38;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.

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FFPE Tumor Tissue Macrodissection: A Technique to Obtain Specific Tumorous Tissue from Unstained Specimens

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FFPE Tumor Tissue Macrodissection: A Technique to Obtain Specific Tumorous Tissue from Unstained Specimens