Dorsal Root Ganglion (DRG) Sample Preparation for Cryosectioning

Begin by post-fixing the DRG tissue dissected from the spinal cord of the anesthetized mouse in 4%formaldehyde for three hours at room temperature. Then, incubate the tissue in 30%sucrose in PBS at four degrees Celsius overnight. Next, prepare the base OCT by adding the OCT compound and covering the top surface of the aluminum block.

Freeze the covered aluminum block at minus 20 degrees Celsius for five to 10 minutes. Cut off the top of the OCT at 30-micrometer thickness using the cryostat, until its surface is as flat as the base OCT. Mark the bottom of the base OCT at the 6:00 position, before taking the aluminum block off the cryostat.

To perform OCT embedment of DRGs, dry the DRG tissue by placing it on a dry Petri dish and moving it from one location to another two or three times with the dry tweezers. Then, place the dry DRG onto the OCT base at the 12:00 position, using dry tweezers. Ensure to keep at least five millimeters distance between the upper-edge of the DRG and the upper-edge of the base OCT, with the dorsal part at the 12:00 position, and the ventral part at the 6:00 position.

Next, add OCT in a round or oval shape to embed the entire DRG tissue with the DRG in the center. Freeze the cover OCT and DRG sample at minus 20 degrees Celsius for five minutes. Once done, cut off the top of the cover OCT at a 30-micrometer thickness until the DRG is visible.