A simple and reliable pretreatment protocol facilitates fluorescent in situ hybridisation on tissue microarrays of paraffin wax embedded tumour samples

Abstract

Aims: To describe a robust pretreatment protocol for preparing paraffin wax embedded tissues on tissue microarrays for fluorescence in situ hybridisation (FISH). The newly developed pretreatment protocol described here was compared with the commonly used sodium thiocyanate based protocol and two different heating methods used in standard antigen unmasking protocols for immunohistochemistry (pressure cooking and microwaving in citrate acid buffer).

Methods: Dewaxed tissue sections were incubated in 10mM citric acid buffer at 80 degrees C for 30 minutes to two hours, followed by a short pepsin digestion (1-5 mg/ml). Pretreated tissues were co-denatured with DNA probes at 80 degrees C for 10 minutes, followed by hybridisation at 37 degrees C for 48-72 hours.

Results: The three protocols using citrate acid buffer produced FISH signals with superior signal to noise ratios compared with sodium thiocyanate pretreatment. Most importantly, the best tissue attachment was achieved using the newly developed pretreatment protocol: on tissue microarrays less than 1% of cores were lost. To date, a total of 30 probes have been successfully hybridised on to breast tissue and multi-tissue microarrays.

Conclusion: This pretreatment protocol is easy, reproducible, and facilitates FISH on tissue microarrays, with potential for widespread application in cancer research.

Figure 1

A flow chart comparing the steps in the pretreatment of paraffin wax embedded tissue sections using either sodium thiocyanate (NaSCN) or citric acid buffer. EtOH, ethanol; dH₂O, distilled water; SSC, saline sodium citrate; RT, room temperature; mins, minutes.

Figure 2

Comparison of the effects of two different pretreatment protocols on the integrity of the tissue cores on a tissue microarray (TMA) using positively charged slides. The sections were treated with 10mM citrate acid buffer (pH 6.0) at 80°C for two hours (left) or 1M sodium thiocyanate at 80°C for 30 minutes (right). Both TMAs were treated with 5 mg/ml pepsin at 37°C for 15 minutes. The sections were stained with haematoxylin and eosin.

Figure 3

(A–D) Assessment of the robustness of the citrate acid buffer/two hour pretreatment protocol in different tissue types: (A) colon, (B) lung, (C) lymph node, and (D) prostate. The following LAVysion probes were used: EFGR–Spectrum Red, c-MYC–Spectrum Gold, chromosome 5p15.2 and centromere of chromosome 6 (CEP6)–Spectrum Aqua. Amplification of EFGR can be seen in the lung tumour (B). (E,F) Assessment of the robustness of the citrate acid buffer/two hour pretreatment protocol using commercial directly labelled probes and in house probes. (E) LAVysion probes; (F) CCDN1 (cyclin D1)–Spectrum Orange and in house labelled GARP–digoxigenin–fluorescein isothiocyanate. The sample shown here is a core from the breast tumour tissue microarray, which is aneuploid (four to six copies) and has a massive amplification on chromosome 11q13 encompassing CCDN-1 and GARP.