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

프로토콜

1. Scraping the Slides

1. 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).
2. Fill a 1.5 mL single-use polypropylene tube with 80 µL of digestion/lysis buffer (see Table of Materials).
3. Put a clean pipette tip into the buffer.
4. Identify cancer tissues of the tumor area most suitable for macrodissection according to the appropriate H&E-stained section.
5. Macrodissect cancer tissue based on the marked H&E-stained section.
   1. 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.
   2. 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.
6. Repeat step 1.5 with the rest of the slides.
7. 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.
8. Add 20 µL of a subtilisin-related serine protease to the vial and gently flick to mix (see Table of Materials).
9. Place the vial in a 55 °C heat block for at least 4 h or overnight. Make sure to slightly vortex after 2 h.

자료

Name	Company	Catalog Number	Comments
10% Sodium dodecyl sulfate (SDS)	Quality Biological	351-032-721 EA	Step 2.1.
DEPC-treated water	Quality Biological	351-068-131	Step 2.1.
Ethylenediaminetetraacetic acid (EDTA)	Corning	46-034-CL	Step 2.1.
Tris hydrochloride (Tris-HCL) 2 M pH 8.8	Quality Biological	351-092-101	Step 2.1.
Proteinase K	New England Biolabs	P8107S	Step 2.8.
Single-use polypropylene (Eppendorf) tube	Eppendorf	24533495	Step 2.5.2.