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

ta en bloc. Transect the heart at the lower poles of the atria and perpendicular to them. This plane is parallel to that of the aortic valve.
  • Place the upper half on the heart (containing the atria) in an embedding mold filled with OCT cryoembedding medium, and allow it to infiltrate into the cavity of the heart.
  • Position the heart flat with the cut surface on the bottom of the embedding mold. Fill the embedding mold with OCT and freeze on dry ice.
  • Store specimens at 80 C.
  • Section tissue blocks at 6 m thickness and mount on positively charged Color Frost Plus slides.

  • B. Immunodetection of CD68+ Macrophages for LCM.

    Standard immunohistochemical staining protocols usually require prolonged incubation periods in aqueous media, which results in significant degradation of RNA. To overcome this limitation, a modified rapid immunostaining protocol was developed for macrophage-specific CD68/Macrosialin that does not significantly affect RNA yields (<12% reduction of total RNA in immunostained versus non-immunostained whole tissue sections) (11).

    1. Allow frozen sections to air dry for 1 min and then fix in 70% ethanol for 15 sec followed by cold acetone for 5 min.
    2. Incubate slides with anti-mouse CD68/macrosialin IgG (1:10 dilution) in 4% goat blocking serum (made in PBS) supplemented with SuperaseIn RNase inhibitor (0.4U/l) for 1 min at room temperature (RT).
    3. Rinse slides in PBS 3 times for 5 min each.
    4. Incubate slides with biotinylated rabbit-anti-rat IgG mouse-adsorbed secondary antibody (20g/ml final) in 4% goat blocking serum supplemented with SuperaseIn RNase inhibitor (0.4U/l) for 1 min at RT.
    5. Rinse slides in PBS 3 times for 5 min each.
    6. Incubate slides with Vectastain ABC Alk-Phos
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