( e. PCR was performed using the Applied ...)Laser Capture Microdissection (LCM) and Expression Profiling of Long-Range Projection Neurons

2. Amplified DNA was purified and cloned into the pCR II-TOPO vector (Invitrogen). The identity of each clone was confirmed by DNA sequencing.

3. Antisense probes were generated by restriction enzyme digestion and transcription from the T7 promoter in the presence of P³³ UTP.

4. Fresh frozen sections of 6-12 m in thickness were thawed and fixed in 4% fresh, cold formaldehyde, followed by a wash in DEPC-treated PBS. The sections were aceylated for 5 minutes in 0.1 M triethanolamine with freshly added acetic anhydride, and after a final wash in PBS the sections were dehydrated in graded ethanol solutions: 35%, 50%, 75%, and 100%.

5. P³³ labeled probe at 1million cpm was added to each section. The sections were coverslipped and submerged overnight in 60^oC hot Mineral oil to avoid evaporation. The next day the sections were dipped 10 times in chloroform and de-coverslipped in 50C warm 2x SSC buffer. After a 1 h wash in 2xSSC, non-specifically bound probe was digested in RNase buffer with 20g/mL RNase A for 15 min. A final wash was performed for 1h in 0.1x SSC, and the sections were dehydrated in 30%, 75%, and 95% ethanol containing 300mM ammonium acetate.

6. In situ hybridizations were visualized on a phosphorimager at low resolution prior to coating with photographic emulsion. The sections were exposed to an X-ray film overnight and then dipped into x-ray film emulsion (Kodak diluted 1:1 with water). The slides were exposed in the dark 2.5-5 weeks (depending on the expected abundance of the gene of interest) and developed as per manufacturers instructions (Kodak).

7. The clustering of silver grains was analyzed under brightfield conditions and compared to the pattern of fluorescent trace
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