Sample preparation from paraffin-embedded tissue specimens for laser scanning cytometric DNA analysis

✍ Scribed by Tomomi Kamada; Kohsuke Sasaki; Tatsuo Tsuji; Takeshi Todoroki; Masahiro Takahashi; Akira Kurose

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 220 KB
• Volume: 27
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/(sici)1097-0320(19970301)27:3<290::aid-cyto12>3.0.co;2-o

✦ Synopsis

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-mm 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°C and then 0.1% RNase for 10 min at room temperature. The slides were dipped in propidium iodide (25 mg/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.