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

עכבר שורש גבי גנגליון Cryosectioning

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.

מחבר זרקור: הליך עבור עכבר שורש גבי גנגליון Cryosectioning  

הליך עבור עכבר שורש גבי גנגליון Cryosectioning

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  

גנגליון שורש גבי (DRG) הכנת דגימה עבור Cryosectioning

התחל על ידי קיבוע לאחר קיבוע רקמת DRG שנותחה מחוט השדרה של העכבר המורדם בפורמלין 4% למשך שלוש שעות בטמפרטורת החדר. לאחר מכן, לדגור על הרקמה ב-30% סוכרוז ב-PBS בארבע מעלות צלזיוס למשך הלילה. לאחר מכן, הכינו את OCT הבסיס על ידי הוספת תרכובת OCT וכיסוי המשטח העליון של בלוק האלומיניום.מקפיאים את גוש האלומיניום המכוסה במינוס 20 מעלות צלזיוס למשך חמש עד 10 דקות. חתכו את החלק העליון של ה-OCT בעובי של 30 מיקרומטר באמצעות הקריוסטט, עד שפני השטח שלו שטוחים כמו ה-OCT הבסיסי. סמנו את החלק התחתון של ה-OCT של הבסיס במצב 6:00, לפני שאתם מורידים את בלוק האלומיניום מהקריוסטט.כדי לבצע הטמעת OCT של DRGs, יבש את רקמת DRG על ידי הנחתה על צלחת פטרי יבשה והעברתה ממקום אחד למשנהו פעמיים או שלוש עם הפינצטה היבשה. לאחר מכן, הניחו את ה-DRG היבש על בסיס OCT במצב 12:00, באמצעות פינצטה יבשה. הקפידו לשמור על מרחק של לפחות חמישה מילימטרים בין הקצה העליון של DRG לבין הקצה העליון של ה-OCT הבסיסי, כאשר החלק הגבי במצב 12:00 והחלק הגחוני במצב 6:00.לאחר מכן, הוסף OCT בצורה עגולה או אליפסה כדי להטביע את כל רקמת DRG עם DRG במרכז. הקפיאו את דגימת OCT ו-DRG בטמפרטורה של מינוס 20 מעלות צלזיוס למשך חמש דקות. לאחר שתסיים, חתכו את החלק העליון של הכיסוי OCT בעובי של 30 מיקרומטר עד שה-DRG יהיה גלוי.

dorsal-root-ganglion-drg-sample-preparation-for-cryosectioning

Research

JoVE Journal

Neuroscience

Dorsal Root Ganglion (DRG) Sample Preparation for Cryosectioning

641 Views.  Capital Medical University. התחל על ידי קיבוע לאחר קיבוע רקמת DRG שנותחה מחוט השדרה של העכבר המורדם בפורמלין 4% למשך שלוש שעות בטמפרטורת החדר. לאחר מכן, לדגור על הרקמה ב-30% סוכרוז ב-PBS בארבע מעלות צלזיוס למשך הלילה. לאחר מכן, הכינו את OCT הבסיס על ידי הוספת תרכובת OCT וכיסוי המשטח העליון של בלוק האלומיניום....

Video: 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 inflammatory and neuropathic pain, itch, as well as other peripheral neurological conditions. However, it remains a challenge to consistently obtain high-quality, intact, and flat cryostat sections onto glass slides because of the tiny sample size of the DRG tissue. So far, there is no article describing an optimal protocol for DRG cryosectioning. This protocol presents a step-by-step method to resolve the frequently encountered difficulties associated with DRG cryosectioning. The presented article explains how to remove the surrounding liquid from the DRG tissue samples, place the DRG sections on the slide facing the same orientation, and flatten the sections on the glass slide without curving up. Although this protocol has been developed for cryosectioning the DRG samples, it can be applied for the cryosectioning of many other tissues with a small sample size.

Presented here is the development for consistently acquiring high-quality dorsal root ganglion cryostat sections.

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of ...

Begin by post-fixing the DRG tissue dissected from the spinal cord of the anesthetized mouse in 4%formaldehyde for three hours at room temperature. Then, incubate the tissue in 30%sucrose in PBS at four degrees Celsius overnight. Next, prepare the base OCT by adding the OCT compound and covering the top surface of the aluminum block.Freeze the covered aluminum block at minus 20 degrees Celsius for five to 10 minutes. Cut off the top of the OCT at 30-micrometer thickness using the cryostat, until its surface is as flat as the base OCT. Mark the bottom of the base OCT at the 6:00 position, before taking the aluminum block off the cryostat.To perform OCT embedment of DRGs, dry the DRG tissue by placing it on a dry Petri dish and moving it from one location to another two or three times with the dry tweezers. Then, place the dry DRG onto the OCT base at the 12:00 position, using dry tweezers. Ensure to keep at least five millimeters distance between the upper-edge of the DRG and the upper-edge of the base OCT, with the dorsal part at the 12:00 position, and the ventral part at the 6:00 position.Next, add OCT in a round or oval shape to embed the entire DRG tissue with the DRG in the center. Freeze the cover OCT and DRG sample at minus 20 degrees Celsius for five minutes. Once done, cut off the top of the cover OCT at a 30-micrometer thickness until the DRG is visible.

Cryosectioning of the Dorsal Root Ganglion (DRG)

Begin the cryosectioning by holding the optimal cutting temperature DRG section at the bottom, using a small paint brush, pre-chilled at 20 degrees Celsius. Gently touch the bottom of the section using the end of a room temperature tweezer so that it sticks to the platform surface. Next, slowly place a charged glass slide over the section.As soon as the section sticks to the slide, gently pull the slide backward. Follow the same process to get more DRG sections onto the slide without overlapping the sections. The proper placement of the DRG sections on the glass slides is seen where the tissues are not overlapping the DRG section and the OCT of other DRG sections.The confocal fluorescence microscopy image demonstrated the quality of the DRG sections obtained with this protocol. With this technique, the immunohistochemistry studies of DRG can be performed using different antibodies.