Practical histological microdissection for PCR analysis

J Pathol. 1996 May;179(1):121-4. doi: 10.1002/(SICI)1096-9896(199605)179:1<121::AID-PATH536>3.0.CO;2-D.


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.

MeSH terms

  • Alleles
  • Base Sequence
  • Chromosome Deletion
  • Colon / cytology
  • Colorectal Neoplasms / genetics
  • Dissection / methods*
  • Humans
  • Molecular Sequence Data
  • Neoplasms / genetics*
  • Polymerase Chain Reaction / methods*