Laser Capture Microdissection of Mammalian Tissue

Published 10/01/2007
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Biology

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Edwards, R. A. Laser Capture Microdissection of Mammalian Tissue. J. Vis. Exp. (8), e309, doi:10.3791/309 (2007).

Abstract

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Disclosures

The authors have nothing to disclose.

Comments

2 Comments

  1. Hi,I am facing problem with RNA isolation of microdissected slides. Frist I staine sections using std protocol of H&E stain (we filter it using Nalgene filter) and then do dissection for 45 minutes, cutting more than 1500 cells. Later RNA is isolated using RNAeasy Micro kit (qiagen). My problem is cutting cells (objects) less than 500 is not detected by Agilent bioanalyser pico chip. And RNA concentratin is too low (1400pg/14ul in 700 cells) to be detected in bioanalyser. Can u suggest me what should I do to enhance RNA integrity number (RIN), ration of ²8s/18s as well as concentration of it. I wonder how to detect RNA in sample having ²0-50 cells  only. Any suggestion at this point will be greately appreciated.Thanks in advanceRatan 

    Reply
    Posted by: Anonymous
    March 20, 2008 - 12:24 AM
  2. If you want to cut out realy low numbers of cells you shouldnt use Leica. Such small areas will never fall down into your cap. It's good for large and heavy areas but not for light and small ones.
    Use a Laser Capture instrument from Carl Zeiss or Molecular Machines & Industries instead. They are best for small samples.

    Reply
    Posted by: Anonymous
    March 30, 2012 - 9:15 AM

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