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

Kryosektion des Spinalwurzelganglion der Maus

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 im Rampenlicht: Ein Verfahren zur Kryosektion des Spinalganglion der Maus  

Ein Verfahren zur Kryosektion von Spinalganglien der Maus

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  

Probenvorbereitung für das Spinalganglion (DRG) für die Kryosektion

Beginnen Sie mit der Nachfixierung des DRG-Gewebes, das aus dem Rückenmark der anästhesierten Maus präpariert wurde, in 4%igem Formaldehyd für drei Stunden bei Raumtemperatur. Dann wird das Gewebe in 30%iger Saccharose in PBS bei vier Grad Celsius über Nacht inkubiert. Bereiten Sie als Nächstes das Basis-OCT vor, indem Sie die OCT-Mischung hinzufügen und die Oberseite des Aluminiumblocks abdecken.Den abgedeckten Aluminiumblock bei minus 20 Grad Celsius für fünf bis 10 Minuten einfrieren. Schneiden Sie die Oberseite des OCT mit dem Kryostaten mit einer Dicke von 30 Mikrometern ab, bis seine Oberfläche so flach ist wie das Basis-OCT. Markieren Sie die Unterseite des Basis-OCT an der Position 6:00, bevor Sie den Aluminiumblock vom Kryostaten entfernen.Um eine OCT-Einbettung von DRGs durchzuführen, trocknen Sie das DRG-Gewebe, indem Sie es auf eine trockene Petrischale legen und zwei- bis dreimal mit der trockenen Pinzette von einer Stelle zur anderen bewegen. Legen Sie dann das trockene DRG mit einer trockenen Pinzette in der 12:00-Position auf die OCT-Basis. Achten Sie darauf, mindestens fünf Millimeter Abstand zwischen dem oberen Rand des DRG und dem oberen Rand des Basis-OCT einzuhalten, wobei sich der dorsale Teil in der 12:00-Position und der ventrale Teil in der 6:00-Position befindet.Als nächstes fügen Sie OCT in runder oder ovaler Form hinzu, um das gesamte DRG-Gewebe mit dem DRG in der Mitte einzubetten. Die OCT- und DRG-Probe für fünf Minuten bei minus 20 Grad Celsius einfrieren. Wenn Sie fertig sind, schneiden Sie die Oberseite der Abdeckung OCT mit einer Dicke von 30 Mikrometern ab, bis das DRG sichtbar ist.

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 ...