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

The video outlines a procedure for processing DRG tissue from a mouse's spinal cord. It details steps for fixing the tissue in formaldehyde, embedding it in OCT compound, and using a cryostat to cut the tissue into 30-micrometer slices for further analysis, ensuring proper orientation and embedding techniques.

dorsal root ganglion (drg) sample preparation for cryosectioning

Cryosectionnement du ganglion de la racine dorsale de souris

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.

Pleins feux sur l’auteur : Une procédure de cryosection du ganglion de la racine dorsale de souris  

Une procédure pour la cryosection du ganglion de la racine dorsale de la souris

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 Dorsal Root Ganglion DRG Sample Preparation for Cryosectioning at JoVE.com

Dorsal Root Ganglion (DRG) Sample Preparation for Cryosectioning  

Préparation d’échantillons de ganglion de la racine dorsale (DRG) pour la cryosection

Commencez par post-fixer le tissu DRG disséqué de la moelle épinière de la souris anesthésiée dans du formaldéhyde à 4 % pendant trois heures à température ambiante. Ensuite, incubez le tissu dans du saccharose à 30 % dans du PBS à quatre degrés Celsius pendant la nuit. Ensuite, préparez l’OCT de base en ajoutant le composé OCT et en recouvrant la surface supérieure du bloc d’aluminium.Congelez le bloc d’aluminium recouvert à moins 20 degrés Celsius pendant cinq à 10 minutes. Coupez le haut de l’OCT à 30 micromètres d’épaisseur à l’aide du cryostat, jusqu’à ce que sa surface soit aussi plane que l’OCT de base. Marquez le bas de l’OCT de base à la position 6 :00, avant de retirer le bloc d’aluminium du cryostat.Pour effectuer l’enrobage OCT des DRG, séchez le tissu DRG en le plaçant sur une boîte de Pétri sèche et en le déplaçant d’un endroit à un autre deux ou trois fois avec la pince à épiler sèche. Ensuite, placez le DRG sec sur la base OCT à la position 12 :00, à l’aide d’une pince à épiler sèche. Assurez-vous de garder une distance d’au moins cinq millimètres entre le bord supérieur du DRG et le bord supérieur de l’OCT de base, avec la partie dorsale à la position 12h00 et la partie ventrale à la position 6h00.Ensuite, ajoutez l’OCT dans une forme ronde ou ovale pour enfoncer l’ensemble du tissu DRG avec le DRG au centre. Congeler l’échantillon OCT et DRG de couverture à moins 20 degrés Celsius pendant cinq minutes. Une fois cela fait, coupez le haut de la couverture OCT à une épaisseur de 30 micromètres jusqu’à ce que le DRG soit visible.

Research

JoVE Journal

Neuroscience

Dorsal Root Ganglion (DRG) Sample Preparation for Cryosectioning

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning

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