Laser Capture Microdissection of muscle fiber populations and expression analysis by RT-PCR ( . The muscle fiber populations in EOM a...)

Laser Capture Microdissection of muscle fiber populations and expression analysis by RT-PCR

. The muscle fiber populations in EOM and limb muscle were distinguished by a combination of the histochemical acetylcholine esterase stain by Karnowsky and Roots⁵ and immunohistochemical staining with a mouse-monoclonal slow myosin heavy chain (MyHC) antibody (NOQ7-5-4D)⁶. SIFs show a large, C-shaped neuromuscular junction after acetylcholine esterase staining and do not react with the anti-slow MyHC antibody. The neuromuscular junction of MIFs is smaller and more circular and all MIFs are positive for slow MyHC by immuno-staining. Since mRNA is sensitive to degradation, total time from thawing the slides to dehydration by xylenes was reduced to 32 minutes by combining the histochemical and immunohistochemical reactions and the direct coupling of the antibody to Alexa Fluor 488 by Molecular Probes Zenon^TM technology.

This protocol describes a way to isolate and visually distinguish muscle fiber population from two different muscle allotypes and characterize different muscle fibers by multiple RT-PCRs.

Equipment and Reagents
This protocol requires the following reagents:
◊ RNase Away (Invitrogen, Cat. # 10328-011)
◊ Zenon^TM Alexa Fluor 488 Mouse IgG1 Labeling Kit (Molecular Probes, Cat. # Z-25002)
◊ PicoPure^TM RNA Isolation Kit (Arcturus, Cat. # KIT0204)
◊ RiboAmp^TM RNA Amplification Kit (Arcturus, Cat. # KIT0201)
◊ Slow Myosin MyHC Antibody NOQ7-5-4D
◊ PCR Primer (Invitrogen, custom-made)
◊ Goat Serum (Sigma, Cat. # G-9023)
◊ Ethanol (Pharmaco, Cat. # 111ACS200)
◊ Hydranal-Xylenes (Riedel-de Haen, Cat. # 37866)
◊ 2-Methylbutane (Fisher, Cat. # A-03551-4)
◊ Colorfrost /Plus Microscope Slides (Fisher
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( . The muscle fiber populations in EOM a...)Laser Capture Microdissection of muscle fiber populations and expression analysis by RT-PCR