( ◊ Disposable gloves ...)Laser Capture Microdissection (LCM) and Expression Profiling of Long-Range Projection Neurons

> ◊ Disposable gloves
◊ Ethanol for freezing brain
◊ Dry ice
◊ Cryostat
◊ Thermal cycler
◊ Microarray Scanner
◊ Microarray analysis software (e.g. GeneTraffic Duo, Affymetrix)
◊ Darkroom
◊ Applied Biosystems 7900HT Fast Real-Time PCR System

RNase-free technique
1. All reagents are purchased and stored under RNase-free conditions.

2. Gloves should be frequently changed, and all surfaces of equipment wiped down with RNase AWAY prior to performing any procedure.

3. Cryostat-cut sections are kept in a box at -80 until ready for LCM. One or two sections at a time are thawed and dehydrated for microdissection.

4.It is very important to keep the time between thawing of each section to the time the captured cells are in extraction buffer as brief as possible, preferably under 45 minutes. Attempting to capture as many cells as will fit on a cap without respect to time will result in degraded RNA from the earliest captures, and is not recommended.

Method
A. Animals, surgery, and tissue processing
1. 1. Adult zebra finches (Taeniopygia guttata) ranging in age from 90 days to 4 years were anesthetized in preparation for surgery, and placed in the stereotaxic apparatus. Fluorescent cholera toxin B (CTB) labeled in red or green was injected into nucleus RA or Area X according to standard stereotaxic coordinates. The anesthetic dosages and stereotaxic coordinates were varied depending on the species of lab animal used. A total of 160 nanoliters of a 2% solution of CTB was injected into each nucleus RA, which has a volume of about 0.25 mm3. A total of 240 nanoliters of CTB was injected into Area X, which has a volume of about 1.5 mm3. These injections were made in even volumes,
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