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Methods for sagittal atlas of the 3 day postnatal rat thoracic spinal cord

A P3 (three day old) rat was used to create the sections. The spinal cord was then extracted and cryoprotected in 30% sucrose in phosphate buffer. The cord was kept in this solution for 24 to 48 hours on a shaker at room temperature. It was then blocked, cut on a microtone into seventy micron sagittal sections, and mounted on subbed slides. The slides were allowed to dry overnight.

Non-phosphorylated, neurofilament proteins were labeled immunohistochemically. Slides were washed with physiological phosphate buffered saline (PBS) three times for five minutes each. The sections were blocked (3% bovine serum albumin and 0.2% triton-X 100 in PBS) and placed on the shaker for two hours at room temperature. They were then transferred to an SMI32 monoclonal antibody (Covance, Emeryville, CA, SMI-32P) overnight. Sections were washed with PBS and a secondary antibody, goat antimouse-CYS antibody (Jackson Labs, West Grove, PA, 115-165-008), was applied for two hours. Sections were washed with PBS three times at five minutes each and allowed to dry at room temperature. Coverslips were mounted using Krystalon (EMD Chemicals Inc., in Gibbstown, NJ).

The sections were photographed with a 10X objective on a Nikon Eclipse microscope using a Spot camera and Spot software (Nikon fluorescence BV-2 filter set) at an exposure time of 300 milliseconds and a gain of 4. Because single sections were too large to be photographed in a single frame, , multiple images were taken of each slide and merged using Canon Photostitch. The images were then inverted in Adobe Photoshop (Adobe), and brightness and contrast were adjusted to optimize clarity.

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