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Neuroscience

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning

Published: June 9th, 2023

DOI:

10.3791/65232

1Department of Pain Management, Xuanwu Hospital, Capital Medical University, 2Department of Anesthesia and Perioperative Care, University of California San Francisco, 3Department of Anesthesiology, Sun Yat-Sen University, 4University of Washington
* These authors contributed equally

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 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.

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,

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For the present study, the animal experiments were approved by UCSF Institutional Animal Care and Use Committee and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory animals. Adult, 8-12-week-old C57BL/6 male and female mice (in-house bred) were used here.

1. DRG sample preparation

  1. Anesthetize the mice with 2.5% Avertin (see Table of Materials). Ensure adequate anesthesia by the lack of response to painful stimulati.......

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The current study collected about 16 continuous, high-quality DRG sections from one mouse L4 DRG. The obtained sections were without any distortion. Figure 1 depicts the step-by-step procedure for the cryosectioning. The removal of extra liquid from the tissue sections is shown in Figure 2. The process of OCT embedment of the tissues is highlighted in Figure 3. Figure 4 shows the proper placement of the.......

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This protocol provides an easy step-by-step procedure for cryostat sectioning of the mouse DRG to obtain high-quality DRG sections on slides reliably.There are four critical steps in this protocol. First, the DRG sample and the tweezers must be dry before putting the DRG sample onto the base OCT. Any liquid surrounding the DRG sample will form an ice shell around it, resulting in DRG sections separating from the OCT and curving up. Second, if the aluminum block does not have a mark, or if the base OCT covers the mark, it.......

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None.

....

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Name Company Catalog Number Comments
Avertin Sigma-Aldrich T48402-25G Anesthetize animal
Epredia Cryotome Cryostat Cryocassettes, 25 mm dia. Crosshatched Fisherbrand 1910 Hold the OCT section at the bottom 
Ergo Tweezers Fisherbrand S95310 Using the end of a tweezer to gently touch the bottom (6 o’clock) of the section so that it sticks to the platform surface to prevent the section from curving back in a roll 
Fisherbrand Superfrost Plus Microscope Slides Fisherbrand 1255015 To collect the DRG section 
Marking pens Fisherbrand 133794  Mark the orientation of base OCT
Scigen Tissue-Plus O.C.T. Compound Fisherbrand  23730571 Embedding medium for frozen tissue specimens to ensure optimal cutting temperature (O.C.T.).

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