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

ocol requires the following reagents:

I. CRYOSECTIONING
◊ Isoflurane (Baxter, Cat. #Forane)
◊ Phosphate Buffered Saline (PBS) (Fisher Scientific, Cat. #FLBP399-1)
◊ OCT cryoembedding medium (VWR, Cat. #25608-930)
◊ ColorFrost Plus slides (Fisher Scientific, Cat. #12-550-17)

II. IMMUNOHISTOCHEMISTRY
◊ Normal goat serum (Vector Laboratories, Cat. #S-1000)
◊ Anti-CD68/Macrosialin antibody (Serotec, Cat. #MCA1957S)
◊ SUPERaseIn (Ambion, Cat. #2694)
◊ Biotinylated rabbit-anti-rat IgG mouse-adsorbed secondary antibody (Vector Laboratories, Cat. #BA-4001)
◊ Vectastain ABC Alk- Phosphatase Kit (Vector Laboratories, Cat. #AK-5200)
◊ Vector Red substrate (Vector Laboratories, Cat. #SK-5100)

III. LASER CAPTURE MICRODISSECTION
◊ PixCell II Laser Capture Microdissection Instrument (Arcturus, Cat. #LCM1104)
◊ CapSure Macro LCM caps (Arcturus, Cat. #LCM0211)

IV. RNA EXTRACTION/ ISOLATION AND QUANTIFICATION
◊ PicoPure RNA Isolation Kit (Arcturus, Cat. # KIT0204)
◊ RNAse-Free DNase Set (Qiagen, Cat. #79254)
◊ Ribogreen RNA quantification kit (Molecular Probes, Cat. #R11490) V. REAL-TIME QUANTITATIVE RT-PCR
◊ Gene-specific TaqMan primers and probes (Biosearch Technologies, Cat. # (see Table I)
◊ SuperScript II reverse transcriptase enzyme (Invitrogen, Cat. #18064-014)
◊ 10mM dNTP mix (Invitrogen, Cat. #18427-013)
◊ Taq DNA polymerase (Invitrogen, Cat. #10342-020)
◊ RnaseOut RNase inhibitor (Invitrogen, Cat. #10777-019)
◊ Acetylated BSA (1g/ml) (Promega, Cat. #R9646A)
◊ 50X 5-carboxy-X-rhodamine (ROX) internal reference dye (Invitrogen, Cat.
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