Method

Cytometry (Comm. Clinical Cytometry) 1996; in press

Method to make paraffin-embedded breast and lymph tissue mimic fresh tissue in DNA analysis

W. Roy Overton, Edison Catalano and J. Philip McCoy, Jr

The following is a summary of the method that was found to optimally prepare paraffin-embedded breast and lymph node for DNA analysis:

  1. Three to ten sections of formalin-fixed, paraffin-embedded (FFPE) tissue are cut 50 microns thick and placed in a nylon mesh pouch. The tissue is dewaxed by three sequential 10 minute incubations in 5 ml of Histo-Solv X (xylene substitute) from Curtin Matheson Scientific, Inc. (Houston, TX; Cat. #245-686).
  2. The dewaxed tissue sections are then rehydrated by 10 minute incubations in 5ml of each of the following alcohol solutions: (1) 100% ethanol (2) 100% ethanol (3) 95% ethanol (4) 95% ethanol (5) 70% ethanol (6) 70% ethanol (7) 50% ethanol (8) 50% ethanol and then three sequential, 5 minute incubations in 5ml of deionized water.
  3. The sections are removed from the pouch and manually homogenized in 10 ml of deionized water using a serrated pestle tissue grinder (Thomas Scientific, Swedesboro, NJ) and held overnight in the deionized water.
  4. The specimen is centrifuged^ and resuspended in a 0.5% trypsin solution (pH 7.5) containing 9 mg/ml sodium chloride and 5 mg/ml trypsin IX (Sigma Chemical Co., St. Louis, MO; Cat.# T-0134) (13,000 -20,000 BAEE units/mg) and immediately incubated in a 37°C water bath for EXACTLY 30 minutes. (DO NOT SUBSTITUTE DIFFERENT TRYPSIN.)
  5. It is then centrifuged^ and resuspended in 4 ml of Dulbecco's phosphate-buffered saline (PBS) (Gibco Laboratories, Grand Island, NY) and then heated for 90 min in a 75°C water bath.
  6. After the heat treatment, the specimen is centrifuged^ and resuspended in 4 ml of PBS.
  7. Fresh DNA-diploid control cells (ie: fresh peripheral whole blood or fresh normal tissue) and the dewaxed, rehydrated, FFPE tissues are then stained for DNA content by the following method. Approximately 106 cells are transferred to an empty test tube and another 0.5 x 106 cells are transferred to a test tube containing approximately 0.5 x 106 DNA-diploid control cells. Approximately 106 DNA-diploid control cells are then added to a third tube.
  8. The three tubes are centrifuged^ and the cells in each tube are then stained by resuspending each pellet in 1ml of PI solution.
  9. The cells are incubated in the PI solution at 4°C for 1 to 4 hours before flow cytometric analysis is performed. Just prior to analysis, the samples may be passed through a 40 - 60 µm nylon mesh filter to remove any large clumps of cells or debris.

^ (centrifuged means centrifuged at 500g for 10 min and the supernatant removed)