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


CONCLUSIONS
This pretreatment protocol is easy, reproducible, and facilitates FISH on tissue microarrays, with potential for widespread application in cancer research.
D001943:Breast Neoplasms / Q000235:genetics; D002021:Buffers; D019343:Citric Acid; D002985:Clinical Protocols; D005260:Female; D006801:Humans; D017403:In Situ Hybridization; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D009369:Neoplasms / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D016612:Paraffin Embedding; D010434:Pepsin A; D015203:Reproducibility of Results; D013861:Thiocyanates
D002021:Buffers; D013861:Thiocyanates; D019343:Citric Acid; C024553:sodium thiocyanate; D010434:Pepsin A