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

Criosección de ganglios de la raíz dorsal de ratón

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 destacado: Un procedimiento para la criosección de ganglios de la raíz dorsal de ratón  

Un procedimiento para la criosección de ganglios de la raíz dorsal de ratón

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  

Preparación de muestras de ganglios de la raíz dorsal (DRG) para crioseccionamiento

Comience fijando el tejido DRG diseccionado de la médula espinal del ratón anestesiado en formaldehído al 4% durante tres horas a temperatura ambiente. A continuación, incubar el tejido en sacarosa al 30% en PBS a cuatro grados centígrados durante la noche. A continuación, prepare la OCT base agregando el compuesto OCT y cubriendo la superficie superior del bloque de aluminio.Congele el bloque de aluminio cubierto a menos 20 grados centígrados durante cinco a 10 minutos. Corte la parte superior de la OCT con un grosor de 30 micrómetros con el criostato, hasta que su superficie sea tan plana como la OCT base. Marque la parte inferior de la OCT base en la posición 6:00, antes de quitar el bloque de aluminio del criostato.Para realizar la inclusión OCT de los GRD, seque el tejido de los GRD colocándolo sobre una placa de Petri seca y moviéndolo de un lugar a otro dos o tres veces con las pinzas secas. A continuación, coloque el DRG seco sobre la base OCT en la posición 12:00, utilizando unas pinzas secas. Asegúrese de mantener una distancia de al menos cinco milímetros entre el borde superior del GRD y el borde superior de la OCT base, con la parte dorsal en la posición 12:00 y la parte ventral en la posición 6:00.A continuación, agregue OCT en forma redonda u ovalada para incrustar todo el tejido DRG con el DRG en el centro. Congele la muestra de OCT y DRG de cobertura a menos 20 grados centígrados durante cinco minutos. Una vez hecho esto, corte la parte superior de la tapa OCT con un grosor de 30 micrómetros hasta que el DRG sea 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 ...