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Laser Capture Microdissection (LCM) for the Analysis of Macrophage Gene Expression from Atherosclerotic Lesions

zyme completely inhibits the reaction and does not produce any visible staining.
  • It is important that the alcohols and the xylenes be prepared fresh and not be reused since insufficient dehydra- tion will dramatically affect the ability to perform successful LCM.
  • RNA extraction of the laser captured material yielded 3.5 ng from 30 6 m thick sections, equivalent to volume of 1.35 x 106 m3 of CD68+ immunos taining).
  • To generate the standard curves for CD68 and α-actin, total RNA was prepared from thioglycol late-elicited intraperitoneal macro- phages and cultured primary aortic smooth muscle cells.
  • The probe, labeled at the 5 and 3 ends with 6-carboxyfluorescein (6-FAM) reporter and tetra methyl-6-carboxyrhodamine (TAMRA) quencher, respectively, is hydrolyzed by the 5 exonuclease activity of Taq DNA polymerase, causing an increase in fluorescent signal that is measured in real-time after each cycle of PCR amplification. Standard curves were constructed by plotting log10 RNA starting quantity vs. cycle threshold (Fig. 3A). On the basis of appropriate serially diluted standard RNA, the amount of input standard RNA yielding the same amount of PCR product measured from an unknown sample was calculated.

  • Method
    A. Animals and Tissue Processing

    1. Apolipoprotein E-/- mice (Jackson Laboratories), a standard model of human atherosclerosis, are fed a standard chow diet for 20 weeks.
    2. Sacrifice mice by exsanguination (under general anesthesia with isoflurane) by intra-vascular perfusion with phosphate buffered saline (PBS). The thorax is opened and a 21-gauge cannula inserted to the left ventricle.
    3. Incise the right atrium to allow efflux of blood. Perfuse at physiological pressure (100mmHg) with PBS.
    4. Remove heart and aor
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