Print version ISSN 1415-4757
Genet. Mol. Biol. vol. 21 n. 4 São Paulo Dec. 1998
A rapid technique for analysis of formalin-fixed, paraffin-embedded tissues by fluorescent in situ hybridization with alpha-satellite probes
Nilce Barril, Andréa Borduchi Carvalho-Salles and Eloiza Helena Tajara
Departamento de Biologia, Instituto de Biociências, Letras e Ciências Exatas de São José do Rio Preto, IBILCE, UNESP, Caixa Postal 136, 15054-000 São José do Rio Preto, SP, Brasil. Send correspondence to E.H.T.
We describe a rapid procedure for preparing archival tissues for interphase FISH analysis. The present protocol differs from others previously described because it allows the obtention of nuclei in satisfactory number and quality without using special equipments, adhesive-treated slides or solutions for chromatin decondensation. The method is of low cost and useful for retrospective analyses of formalin-fixed, paraffin-embedded samples.
Fluorescence in situ hybridization (FISH) is a reliable technique for detecting chromosomal aberrations and has found increasing applications in pathology diagnosis and cancer research. Contrary to conventional cytogenetic techniques, FISH can be used to elucidate karyotypic abnormalities in interphase nuclei, avoiding the need to culture the cells. Interphase cytogenetics is especially important for retrospective analysis of rare tumors, tissues with low mitotic index or comparison of samples from patients in different stages of a disease.
We describe here a modified technique previously described by Harris et al. (1995) and Hyytinen et al. (1994) for interphase FISH analysis of tissues that have been fixed in formalin and embedded in paraffin, as follows (The commentaries of other authors are included in parentheses and in italics).
ISOLATION OF NUCLEI FROM TISSUE SECTIONS
Three 30-50-mm tissue sections were deparaffinized with 5 ml of xylene, twice for 10 min each, and washed in 5 ml of 100% ethanol, twice for 10 min each, and 5 ml of deionized water (phosphate-buffered saline) (Harris et al., 1995), twice for 10 min each. Samples were transferred to a Petri dish and, after drying, were minced with scalpel and resuspended in pepsin - Sigma No. P6887 - 5 mg/ml in 0.2 M NaCl, pH 1.5 (pepsin - 0.7 mg/ml in 3% polyethylene glycol, pH 1.5) (Harris et al., 1995). The mixture was transferred to a tube, vortexed vigorously and incubated for one hour at 37°C. The digested material was rinsed three times using 5 ml of 60% acetic acid (rinsed repeatedly through a 45 micro filter with Hank's basic salt solution - HBSS - until the pH in the solution had returned to approximately 7) (Harris et al., 1995; Hyytinen et al., 1994) and centrifuged for 10 min at 1,200 RPM. After the final rinse, the pelleted nuclei were resuspended in 5 ml of 60% acetic acid (HBSS) and dropped onto cold, wet slides (adhesive-treated slides) (Harris et al., 1995; Hyytinen et al., 1994) which were stored at room temperature for 48 h (the slides were fixed in 3:1 methanol/acetic acid for 10 min, incubated in protease K 1 mg/ml in HBSS for 30 min and soak in fresh fix) (Harris et al., 1995).
DENATURATION AND HYBRIDIZATION
Immediately before use in FISH (the slides were incubated in a water bath for 3 min at 90°C in a 50% glycerol/0.1xSSC, pH 7.5 solution) (Hyytinen et al., 1994) cellular DNA was denatured in 70% formamide/2xSSC solution, pH 7.0 (1xSSC is 0.15 M NaCl, 0.015 M Na citrate) for 4 min at 74°C. The slides were dehydrated for 2 min each in 70, 85 and 100% ethanol, followed by treatment with proteinase K - Sigma P-6911 - 8 mg/ml in 20 mM Tris/2 mM CaCl2 buffer, pH 7.5, for three min at 37°C and ethanol dehydration.
The hybridization and the detection procedures were performed with probes from ONCOR and avidin/FITC system from VECTOR and followed the manufacturer's instructions with modifications. Three microliters of the probe to be used and 60 ml of Hybrisol VI buffer - ONCOR No. S1340-3 - were mixed in a 0.5-ml microcentrifuge tube and spun briefly to consolidate the mixture. The hybridization mixture was denatured for 5 min at 74°C, cooled on ice, and applied to denatured cells on slides. A coverslip was added and sealed with rubber cement. An overnight hybridization was carried out in a humid chamber at 37°C. The coverslips were removed and the slides were subsequently washed in 0.5xSSC for 4 min at 74°C, followed by immunostaining with 60 ml of avidin-FITC for 30 min at 37°C. The slides were washed three times for 2 min each in buffer II (0.1 M Tris-HCl, 0.15 M NaCl and 0.05% v/v Tween 20, pH 7.0) at 37°C and FITC signal was amplified using 60 ml of biotinylated anti-avidin antibody, followed by another layer of 60 ml of avidin-FITC. After three buffer II washes (2 min each), the nuclei were counterstained by adding propidium iodade (1 mg/ml) in an antifade solution - Sigma No. 6001. Figure 1 illustrates nuclei obtained through this procedure.
Figure 1 - Photomicrograph of nuclei prepared from a formalin-fixed, paraffin-embedded sample of a thyroid cancer specimen. FISH was done with a chromosome 7 centromeric probe. Nuclei were counterstained with propidium iodide.
This method was developed in order to reduce the costs and the work time and proved to be valuable for interphase cytogenetic analyses of archival rare tumors and tissues with low mitotic index.
The authors thank Dr. James Robert Coleman for reviewing the text and FAPESP, FUNDUNESP, CAPES and CNPq for financial support. Publication supported by FAPESP.
Descrevemos aqui um procedimento rápido para obtenção de núcleos interfásicos a partir de amostras arquivadas que podem ser utilizados para análise citogenética através da técnica de FISH. Este procedimento difere de outros previamente descritos porque permite a obtenção de núcleos em número e qualidade satisfatórios sem a utilização de equipamentos ou lâminas especiais e soluções para descondensação da cromatina. O método é de baixo custo e possibilita estudos retrospectivos de tecidos fixados em formol e emblocados em parafina.
Harris, C., Robinson, T., Michalski, K. and Meisner, L. (1995). Specimen salvage with FISH. App. Cytogenet. 21: 66-70. [ Links ]
Hyytinen, E., VisakorpIi, T., Kallioniemi, A., Kallioniemi, O.-P. and Isola, J.J. (1994). Improved technique for analysis of formalin-fixed, paraffin-embedded tumors by fluorescence in situ hybridization. Cytometry 16: 93-99. [ Links ]
(Received December 30, 1997)