2. After allowing 3-7 days for retrograde transport of the tracers, birds were sacrificed by decapitation and their brains quickly dissected. RNAlater was applied to the surface of the brain overlying HVC within 1 minute of decapitation. Note: This application of RNAlater helps ensure the preservation of RNA profiles representative of the in vivo situation. However, this is not possible for structures buried deep within the brain, as RNAlater does not diffuse more than a few millimeters.
3. Each brain was quickly dissected, placed in OCT in a plastic tissue mold, and rapidly frozen by placing the mold in a slurry of ethanol and dry ice. NOTE: Be careful not to let any ethanol get into the OCT.
4. Brains were sectioned at 8 m thickness onto glass slides using a cryostat. The sections were stored in a slide box at -80C until ready for LCM.
B. Laser capture microdissection
1. Frozen sections were allowed to thaw until frost was seen to retract from the edges of the slide (approximately 30 seconds). Slides were immediately placed in 75% ethanol for 30 seconds for fixation, followed by a 15 second distilled water rinse. NOTE: All alcohols and xylenes should be prepared fresh. Steps using 100% EtOH are particularly sensitive, as small amounts of water at this stage may interfere with microdissection. This lab changes the 100% EtOH at least twice each day, and more often if the LCM pick-up is not optimal.
2. Dehydration proceeded as follows:
75% EtOH--30 sec
95% EtOH--30 sec
100% EtOH--30 sec
Fresh 100% EtOH--1 min
Fresh 100% EtOH--3 min
Fresh Xylene--2 min
Air dry--3 min