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

uantities of the four solutions can be prepared and stored at 4C. But the staining solution has to be prepared fresh since the acetylthiocholine starts to precipitate out of solution after 2 hours.

4. To stain slow MyHC positive fibers rapidly Molecular Probess ZenonTM antibody-labeling technology is used. 20 l of NOQ7-5-4D are mixed with 5 l of Component A (ZenonTM mouse labeling reagent), incubated for 5 minutes. Followed by 5 minutes of incubation with Component B (ZenonTM mouse blocking reagent).

5. After adding Component B to NOQ75-4D, the slide for laser capture microdissection is placed in DEPC-treated water with 2% heat inactivated goat serum for 5 minutes to block non-specific binding sites.

6. The Alexa Fluor 488 conjugated NOQ7-5-4D antibody is diluted 1:40 in the prepared acetylcholine esterase staining solution.

7. The blocked slide is now incubated with the prepared staining solution for 20 minutes.

8. After 20 minutes, the slide is washed twice for 3 minutes in DEPC treated water.

9. The slide is dehydrated in 75%, 90%, and 100% ethanol for 30 seconds respectively, followed by 5 and 7 minutes in fresh xylenes. After 1 minute in a flow hood the slide is ready for laser capture microdissection.

10. The Arcturus PixCell II Laser Capture Microdissection System with fluorescence package is switched on and the blue filter cube is set. The QImaingTM Retiga 1300 cooled mono 12-bit digital camera is activated.

11. The microdissection laser is set to 7 m size, 70 mW power and 850 s pulse duration. The last two values are adjusted to the lowest values possible to still perform laser capture microdissection. This adjustment to a minimal setting enables to actually dissect single muscle fibers.

12. SIFs were dissected based on their large neuromuscular junction stained by Karnowsky and Rootss
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