Sample preparation from paraffin-embedded tissue specimens for laser scanning cytometric DNA analysis.
We have developed a simple, rapid method for isolating cells from a block of formalin-fixed, paraffin-embedded tissue specimen for laser scanning cytometric (LSC) DNA analysis by using a grater. The scraping-like tissue samples were obtained by grating a paraffin-embedded tissue block. The grated samples were collected, put into a small plastic tube, and deparaffinized with xylene. Subsequently, the samples were immersed in 100% ethanol to remove the xylene. After using a syringe with a 26-gauge needle and filtering through 40-microm nylon mesh, the cells suspended in ethanol were dropped directly onto a glass slide. As a result, isolated cells adhered tightly to the glass slide. The slides mounted with isolated cells were treated with 0.1% pepsin in 0.1 N HCl for 1 h at 37 degrees C and then 0.1% RNase for 10 min at room temperature. The slides were dipped in propidium iodide (25 microg/ml) to stain DNA and sealed with nail varnish. The coefficients of variation for histograms were small enough to detect an aneuploid peak close to the diploid peak.
Kamada
T
T
Department of Pathology, Yamaguchi University School of Medicine, Ube, Japan.
Sasaki
K
K
Tsuji
T
T
Todoroki
T
T
Takahashi
M
M
Kurose
A
A
D000782:Aneuploidy; D004273:DNA, Neoplasm / Q000737:chemistry*; D004171:Diploidy; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D007834:Lasers; D016612:Paraffin Embedding / Q000379:methods*; D011419:Propidium / Q000737:chemistry
D004273:DNA, Neoplasm; D011419:Propidium