Single-day FISH procedure for paraffin-embedded tissue sections using a microwave oven

The fluorescence in situ hybridization (FISH) technique is a sensitive method for detecting gene amplification in tumor cells. However, FISH on formalin-fixed and paraffin-embedded tissue sections is time-consuming and labor-intensive (1). Tissue processing and cross-linking fixatives such as formalin may prevent the probe from accessing its target sequence, prolonging the FISH processing time for fixed tissues compared to cells in suspension or frozen material (2,3). For example, the standard FISH procedure for fixed tissue takes about 2 days and contains several acid/chaotrope-based pretreatment steps and an overnight hybridization (www.vysis.com/ PretreatingParaffinSpecimens_32217. asp). Despite its sensitivity and specificity, FISH on paraffin sections is therefore not the method of choice in many clinical and research laboratories. Here we propose a simplified and rapid FISH protocol for fixed tissues using microwave irradiation. Using the commercially available probe mixture of HER-2 LSP (locusspecific probe) and CEP17 (centromere enumeration probe for chromosome 17, α-satellite DNA; Vysis/Abbott, Downers Grove, IL, USA), FISH was conducted on 4 μm thick formalinfixed, paraffin-embedded tissue sections from 9 breast cancer and 7 endometrial cancer cases. Normal human lymphocytes and 4 breast cancer cell lines, all embedded in paraffin, were used as controls for hybridization and scoring efficiency (4). In total, 21 specimens were analyzed. Sections from the same block were treated either with the commonly used FISH procedure developed by Vysis or the new microwave procedure. For each case, an average of 62 (30–173) cells were scored and the mean copy number for each probe, the HER-2/CEP17 ratio, and the proportion of aneusomic cells were compared between the methods (for statistical analysis, see Supplementary Table S1 available online at www.BioTechniques.com). In this study, a Samsung Microwave 1100W (Model no. MW1080STA, with 10 power levels; Samsung Electronics America, Ridgefield Park, NJ, USA) was used. The microwave settings for other brands of microwaves can be determined by measuring the temperatures in the buffer (pretreatment step) and water (hybridization step). The optimal temperatures are written in parentheses after each irradiation step in Protocol 1. Keep in mind that irradiation time must be optimized to prevent the loss of tissue morphology, but the tissue has to be irradiated long enough for the LSP probe to hybridize. The mean copy number results from the two procedures were highly comparable (see Supplementary Table S1). The microwave FISH procedure performed equally well for both nonamplified and amplified cases as well as controls, with two exceptions. In BT474 cells, the mean absolute HER-2 signal per cell ±sd was slightly higher after microwave treatment (34.0 ± 11.0) compared to standard treatment (28.1 ± 10.5), and the difference reached significance (P = 0.002). We ascribe the observed difference to the complex nature of the HER-2 amplification in these cells, rather than to the type of treatment. In BT-474 cells, due to the formation of compact heterogeneous clusters of HER-2 signals, the identification of individual signals can be difficult and may result in slightly variable numbers of the mean HER-2 signals per cell in repetitions (4,5). However, the other FISH parameters showed comparable values, and the results from both treatments meet the criteria for HER-2 amplification status in the BT-474 cells (4,5). The same explanation is applicable to the one cancer case (see Supplementary Table S1, case 14). Our rapid microwave protocol showed high hybridization efficiency and gave us signals that were as bright (Figure 1, B, D, and F) as the traditional method (Figure 1, A, C, and E). In Figure 1, A and B, adjacent tissue sections from the same breast cancer case are shown. The HER-2/CEP17 ratios were 4.2 (standard treatment) and 4.5 (microwave) (P = 0.448). By