Name: Cryosectioning of the Dorsal Root Ganglion (DRG)
Uploaded: 2023-06-09T13:50:00
Description: Watch this Scientific Journal Video about Cryosectioning of the Dorsal Root Ganglion DRG at JoVE.com

The video demonstrates a cryosectioning technique for obtaining discrete dorsal root ganglion (DRG) tissue sections. It details the process of attaching DRG sections to glass slides without overlap, ensuring optimal placement for subsequent immunohistochemistry studies. The effectiveness of the method is confirmed through confocal fluorescence microscopy imaging.

cryosectioning of the dorsal root ganglion (drg)

Mouse Dorsal Root Ganglion Cryosectioning

The dorsal root ganglion (DRG) contains the primary sensory neurons, the tissue macrophages, and the satellite cells that surround the primary sensory neurons1,2,3,4. It is a key anatomic structure in processing innocuous and noxious signals, and plays critical roles in pain, itch, and various peripheral nerve disorders5,6,7,8,9,10,11,12,13. Although several methods have been developed to dissect DRG tissue from the mouse spinal cord14,15,16, cryosectioning of the DRG tissue remains challenging as the DRG tissue is quite small, and cryostat sections of DRG samples tend to curve into rolls, making it difficult to properly transfer the cryostat sections onto glass slides. However, proper cryosectioning of the DRG tissue is crucial for immunohistochemistry studies and the structure of DRG sensory neurons17,18,19,20,21,22,23. Moreover, as single-cell RNA sequencing results have revealed the remarkable heterogeneity of DRG sensory neurons in both humans24 and mice25, proper cryosectioning of DRG tissue is critical for investigating the functional role of different DRG cells in various physiological and pathological conditions.
Although the tissue-clearing technique has been applied to investigate the 3D reconstruction of the DRG26 as an alternative technique of cryosectioning the DRG, the tissue-clearing technique is time- and labor-consuming. In comparison, cryosectioning of the DRG is quick and relatively easy to perform, and thus it remains to be a key technique for immunohistochemistry and structure studies of the DRG and other regions of the central nervous system. However, obtaining high-quality, intact, and flat cryostat sections onto glass slides remains to be a challenge in neuroscience research because of the tiny sample size of tissues, like the DRG and certain brain regions, and there is no article describing the optimal protocol at this point for cryosectioning small-sized tissue samples, such as mouse DRGs.
This protocol provides an easy, step-by-step technique for cryostat sectioning of the mouse DRG to reliably obtain as many high-quality DRG sections on the slides for subsequent DRG studies. While specifically designed for cryosectioning DRG samples, this technique can potentially be used for cryosectioning various other tissues with a small sample size.

Author Spotlight: A Procedure for Mouse Dorsal Root Ganglion Cryosectioning  

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning

It remains a challenge to consistently 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 cryostat 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 cryostat 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 roll, 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.

Watch this Scientific Journal Video about Cryosectioning of the Dorsal Root Ganglion DRG at JoVE.com

Cryosectioning of the Dorsal Root Ganglion (DRG)  

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.

Research

JoVE Journal

Neuroscience

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of ...