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

Kryoseksjonering av mus ryggrot ganglion

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.

Forfatter i søkelyset: En prosedyre for kryoseksjon av ryggrotganglion hos mus  

En prosedyre for mus dorsal root ganglion kryoseksjonering

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  

Dorsal root ganglion (DRG) prøveklargjøring for kryoseksjonering

Begynn med å legge inn DRG-vevet dissekert fra ryggmargen til den bedøvede musen i 4% formaldehyd i tre timer ved romtemperatur. Deretter inkuberer vevet i 30% sukrose i PBS ved fire grader Celsius over natten. Deretter forbereder du basen OCT ved å legge til OCT-forbindelsen og dekke den øverste overflaten av aluminiumsblokken.Frys den dekkede aluminiumsblokken ved minus 20 grader Celsius i fem til 10 minutter. Klipp av toppen av OCT ved 30 mikrometer tykkelse ved hjelp av kryostaten, til overflaten er like flat som basen OCT. Merk bunnen av basen OCT i 6:00-posisjonen, før du tar aluminiumsblokken av kryostaten.For å utføre OCT-innebygging av DRG, tørk DRG-vevet ved å plassere det på en tørr petriskål og flytte det fra ett sted til et annet to eller tre ganger med den tørre pinsetten. Deretter plasserer du den tørre DRG på OCT-basen i 12:00-posisjonen, ved hjelp av tørr pinsett. Sørg for å holde minst fem millimeter avstand mellom øvre kant av DRG og øvre kant av basen OCT, med dorsaldelen i 12:00-posisjonen og den ventrale delen i 6:00-posisjonen.Deretter legger du til OCT i rund eller oval form for å legge inn hele DRG-vevet med DRG i midten. Frys dekselet OCT og DRG-prøven ved minus 20 grader Celsius i fem minutter. Når du er ferdig, kutt av toppen av dekselet OCT med en tykkelse på 30 mikrometer til DRG er synlig.

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 ...