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

Muis Dorsale Wortel Ganglion Cryosectionering

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.

Auteur in de schijnwerpers: een procedure voor cryosectie van het ganglion met dorsale wortel van muizen  

Een procedure voor cryosectie van het ganglion met dorsale wortel van muizen

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  

Dorsale wortelganglion (DRG) monstervoorbereiding voor cryosecties

Begin met het postfixeren van het DRG-weefsel dat uit het ruggenmerg van de verdoofde muis is ontleed in 4% formaldehyde gedurende drie uur bij kamertemperatuur. Incubeer vervolgens het weefsel in 30% sucrose in PBS bij vier graden Celsius 's nachts. Bereid vervolgens de basis OCT voor door de OCT-verbinding toe te voegen en het bovenoppervlak van het aluminium blok te bedekken.Vries het afgedekte aluminium blok vijf tot 10 minuten in bij min 20 graden Celsius. Snijd de bovenkant van de OCT af met een dikte van 30 micrometer met behulp van de cryostaat, totdat het oppervlak net zo vlak is als de basis OCT. Markeer de onderkant van de basis OCT op de 6:00-positie, voordat u het aluminium blok van de cryostaat haalt.Om OCT-inbedding van DRG's uit te voeren, droogt u het DRG-weefsel door het op een droge petrischaal te plaatsen en het twee of drie keer van de ene locatie naar de andere te verplaatsen met het droge pincet. Plaats vervolgens de droge DRG op de OCT-basis op de positie van 12:00 uur, met behulp van een droog pincet. Zorg ervoor dat u ten minste vijf millimeter afstand houdt tussen de bovenrand van de DRG en de bovenrand van de basis-OCT, met het dorsale deel in de 12:00-positie en het ventrale deel in de 6:00-positie.Voeg vervolgens OCT toe in een ronde of ovale vorm om het hele DRG-weefsel in te bedden met de DRG in het midden. Vries het afgedekte OCT- en DRG-monster gedurende vijf minuten in bij min 20 graden Celsius. Als u klaar bent, snijdt u de bovenkant van de hoes af met een dikte van 30 micrometer totdat de DRG zichtbaar is.

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 ...