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

reagent (prepared at least 30 minutes in advance according to manufacturers instructions) for 1 min. at RT (see Special Considerations, Note 1).
  • Rinse slides in 100mM Tris-HCl Buffer pH. 7.5 for 1 min. at RT.
  • Apply Vector Red chromogenic substrate until adequate intensity of staining develops (~5 - 10 min). Immerse slides in RNase-Free water to stop reaction.
  • Sections are counterstained with Harris modified hematoxylin for ~ 5 sec.
  • Sections are subsequently dehydrated in graded ethanols (95% 2X, 1 min each, 100% 3X, 3 min each ) and cleared in xylene (3X, 2 min each). Allow slides to air dry under a vacuum hood for 30 min (see Special Considerations, Note 2).

  • C. LCM and RNA Extraction

    Laser capture was performed under direct microscopic visualization on the CD68positively stained areas by activating a thermoplastic film mounted on optically transparent LCM caps over selected regions. 30 proximal aortic sections from each apolipoprotein E-deficient mouse were microdissected in regions immunostained positive for CD68/Macrosialin and with morphologically-identifiable cells having the characteristic foamy appearance. Approximately 300 laser pulses were performed on each section using the PixCell II LCM Instrument. The following parameters were used: 15m laser diameter, 40 mW power, 3.0 msec duration.



    1. RNA was extracted/isolated using the PicoPure RNA Isolation Kit as per manufacturers instructions.
    2. Examine the surface of the LCM cap under the microscope to ensure complete cell extraction.
    3. To eliminate potential genomic DNA contamination, RNA samples were treated with the DNase (Qiagen) directly onto the column as per manufacturers instructions.
    4. RNA concentra
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    Recommend to use at up to 5000X dilutions for cell culture (e.g. Add 1 ul to 1 ml of culture medium.)...
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