It remains a challenge to constantly obtain high-quality, intact, and flat mouse dorsal root ganglion cryostat sections. The research aims to answer how to acquire ideal mouse dorsal root ganglion crystat section. This protocol provides an easy step-by-step technique for cryostat section of the mouse DRG to high quality, intact, and flat mouse DRG cross-sections.
This presented article explains how to remove surrounding liquid from the mouse DRG sample tissue, placing the DRG sections on the slides facing the same orientation, and flatten the DRG sections without curving up. One major challenge is that the OCT sections of mouse DRG tend to curve into a row, making it difficult to place onto the glass slide. It is also challenging to acquire sufficient sections from one sample due to the small size of the mouse DRG.
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 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 six o'clock 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 o'clock 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 o'clock position and the ventral part at the six o'clock 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 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.
Begin the cryosectioning by holding the optimal cutting temperature DRG section at the bottom using a small paint brush pre-chilled at 20 degrees Celsius. Gently touch the bottom of the section using the end of a room-temperature tweezer so that it sticks to the platform surface. Next, slowly place a charged glass slide over the section.
As soon as the section sticks to the slide, gently pull the slide backward. Follow the same process to get more DRG sections onto the slide without overlapping the sections. The proper placement of the DRG sections on the glass slides is seen where the tissues are not overlapping the DRG section and the OCT of other DRG sections.
The confocal fluorescence microscopy image demonstrated the quality of the DRG sections obtained with this protocol. With this technique, the immunohistochemistry studies of DRG can be performed using different antibodies.
