title: 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.
authors.affiliation: University Department of Pathology, Glasgow Royal Infirmary, U.K.
authors.forename: J J
authors.initials: JJ
authors.lastname: Going
authors.forename: R F
authors.initials: RF
authors.lastname: Lamb
mesh_terms: D000483:Alleles; D001483:Base Sequence; D002872:Chromosome Deletion; D003106:Colon / Q000166:cytology; D015179:Colorectal Neoplasms / Q000235:genetics; D004210:Dissection / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*