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

Crioseccionamento do gânglio da raiz dorsal do rato

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.

Autor em destaque: um procedimento para crioseccionamento do gânglio da raiz dorsal do camundongo  

Um procedimento para criossecção do gânglio da raiz dorsal de camundongos

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  

Preparação de amostra de gânglio da raiz dorsal (DRG) para criossecção

Iniciar pela pós-fixação do tecido DRG dissecado da medula espinhal do camundongo anestesiado em formaldeído a 4% por três horas à temperatura ambiente. Em seguida, incubar o tecido em 30% de sacarose em PBS a quatro graus Celsius durante a noite. Em seguida, prepare a OCT base adicionando o composto OCT e cobrindo a superfície superior do bloco de alumínio.Congele o bloco de alumínio coberto a menos 20 graus Celsius por cinco a 10 minutos. Corte a parte superior do OCT a 30 micrômetros de espessura usando o criostato, até que sua superfície seja tão plana quanto o OCT de base.Para realizar o embutimento de DRGs em OCT, seque o tecido DRG colocando-o em uma placa de Petri seca e movendo-o de um local para outro duas ou três vezes com a pinça seca. Em seguida, coloque o DRG seco na base do OCT na posição 12:00, usando uma pinça seca. Certifique-se de manter pelo menos cinco milímetros de distância entre a borda superior do DRG e a borda superior do OCT de base, com a parte dorsal na posição 12:00 e a parte ventral na posição 6:00.Em seguida, adicione a OCT em uma forma redonda ou oval para incorporar todo o tecido DRG com o DRG no centro. Congele a amostra de OCT e DRG da tampa a menos 20 graus Celsius por cinco minutos. Uma vez feito, corte a parte superior da OCT da tampa em uma espessura de 30 micrômetros até que o DRG esteja visível.

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 ...