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

#12223-012)

The following laboratory equipment is required to complete the protocol:
◊ Disposable gloves
◊ Dry ice
◊ Cryostat
◊ ABI Prism 7700 Sequence Detection System (Applied Biosystems)
◊ RNase AWAY (Fisher Scientific, Cat. #14-375-35)
◊ Nuclease-free pipette tips
◊ MicroAmp optical tubes (Applied Biosystems, Cat. #N801-0933)
◊ MicroAmp optical caps (Applied Biosystems, Cat. #N801-0935)
◊ Nusieve 3:1 agarose (BioWhittaker Molecular Applications)
◊ Immunostaining jars


RNase-free Technique
All reagents were maintained under RNase-free sterile conditions.

  1. Wear disposable gloves and change them frequently.
  2. Pipettmen are wiped with RNase AWAY prior to use according to manufacturers instructions.
  3. Cryosections are temporarily stored in the cryostat and later into a -80 C freezer to prevent RNA degradation.
  4. Use RNase-free solutions (water prepared by treatment with
  5. Concentration of antibodies and duration of incubations have been optimized so as to preserve the quality of RNA and to yield specific staining having a high signal/noise ratio.
  6. Addition of SuperaseIn RNase Inhibitor to the antibody cocktails during immunostaining is recommended as it prevents RNA degradation during the incubation steps. (NOTE: Do not add the SuperaseIn to the ABC Alk-Phos enzymatic reaction as it will completely inhibit the staining).


Special Considerations

  1. We have observed that addition of the SuperaseIn RNase inhibitor to the alka- line phophatase en
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