( ta en bloc. Transect the heart at the l...)Laser Capture Microdissection (LCM) for the Analysis of Macrophage Gene Expression from Atherosclerotic Lesions

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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