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Laser Capture Microdissection of Cells Labeled with Enhanced Green Fluorescent Protein

equence Detection System (Applied Biosystems, Foster City, CA)


Special Considerations
Excessive fixation of the tissue prior to embedding prevents efficient extraction of RNA from captured cells. Illumination of tissue sections under epifluorescence optics should be minimized in order to reduce photo-bleaching and prolong EGFP fluorescence.


Method
CRYOSECTIONING AND LCM
1. Fix freshly dissected tissues in 4% paraformaldehyde in phosphate buffered saline (PBS) for 5 min. Mild fixation by 4% PFA retains EGFP within the cells during embedding and sectioning without affecting subsequent RNA extraction. Wash the tissue twice in PBS for 5 min, then freeze in OCT compound on dry ice.

2. Pre-clean the internal surfaces of the cryostat with 100% ethanol. Perform cryosectioning onto silanized glass slides in a cryostat at 19 C using a fresh disposable low-profile blade under RNase-free conditions. Mount sections onto slides at room temperature but subsequent storage should be inside the cryostat (short term) or at 70 C (long term).

3. Process tissue sections for LCM within 2 days of cryosectioning. Allow mounted tissue sections to thaw for no longer than 20 seconds before beginning dehydration. Dehydrate sections in 70%, 95% and 100% ethanol for 30 seconds each, followed by 5 min in xylene. Air-dry sections for 2 minutes before commencing LCM.

4. Visualize EGFP-expressing cells in the tissue sections using the EGFP-optimized emission filters with an integration setting of 0.12-0.2 using an Arcturus PixCell IIe system equipped with epifluorescence optics.

5. A spot size of 7.5 m with power settings of 60-70 mA and a pulse duration of 1.3 msec ensured efficient capture of cells.

6. The laser spot overwhelms green fluorescence, so comparison of an image map acquired under fluorescence with the live camera image (under brightfiel
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