Practical histological microdissection for PCR analysis

Abstract

Recovery of cells by histological microdissection is increasingly used for analysis by polymerase chain reaction (PCR) or microchemical techniques. This paper describes techniques of histological microdissection. Sections of archival formalin-fixed, paraffin-embedded tissue up to 15 years old were mounted on plain glass slides. Sections 6-7 microns in thickness stained with toluidine blue were dissected under proteinase K buffer solution, using an electrolytically sharpened tungsten needle in a bacteriological loop-holder and a Leitz mechanical micromanipulator (model M). Detached cell groups were recovered in a silicone-coated pipette tip for PCR analysis after digestion in 25-50 microliters of proteinase K (500/ml) in TRIS-HCl buffer (pH 8.3). Consistent amplification and analysis of microsatellite loci were obtained from 2 microliters of crude lysate using 28-30 cycles of PCR incorporating a 32P 5'-end-labelled primer, electrophoresis under denaturing conditions on 6 per cent polyacrylamide gels, and autoradiographic detection.