A Procedure for Mouse Dorsal Root Ganglion Cryosectioning

Liangliang He; Wenxing Zhao; Lingyi Zhang; Maalveka Ilango; Na Zhao; Liqiang Yang; Zhonghui Guan

doi:10.3791/65232

JoVE Journal > Neuroscience

Neurociência

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning

Published: June 09, 2023

doi:

10.3791/65232

Liangliang He*^1,2, Wenxing Zhao*¹, Lingyi Zhang^2,3, Maalveka Ilango^2,4, Na Zhao², Liqiang Yang¹, Zhonghui Guan²

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

Summary

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

Abstract

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.

Introduction

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.

Protocol

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

Anesthetize the mice with 2.5% Avertin (see Table of Materials). Ensure adequate anesthesia by the lack of response to painful stimulation. Perfuse the animals transcardially with 1x phosphate-buffered saline (PBS) followed by 4% formaldehyde, as previously described¹⁴^,¹⁵^,¹⁶.
Dissect DRG tissue from the spinal cord, as previously described¹⁴^,¹⁵^,¹⁶.
Post-fix the dissected DRGs in 4% formaldehyde for 3 h at room temperature.
Put the DRGs in 30% sucrose in PBS at 4 °C overnight.
Preparing the base optimal cutting temperature (OCT)
1. Add the OCT compound (see Table of Materials) to cover the top surface of the aluminum block and freeze at -20 °C for 5-10 min (Figure 1A).
2. Cut off the top of the OCT using the cryostat (see Table of Materials) at 30 µm thickness until its surface is flat as the base OCT (Figure 1B).
3. Make a mark at the bottom (six o'clock position) of the base OCT to mark its orientation, before taking the aluminum block off the cryostat (Figure 1C).
4. In the following steps, keep the mark at the six o'clock position to maintain the same orientation, which can lead to obtaining more sections of the DRG sample.
  NOTE: If the orientation is lost and the DRG sample is cut at a different angle, the DRG sample cannot be cut completely from top to bottom, which will leave some samples left out on the base OCT and wasted, as the base OCT will be encountered while trying to cut the DRG sample (Figure 1C).
Performing OCT embedment of DRGs
1. Dry the DRG tissue before putting it on the OCT.
  1. Before adding the DRG onto the block, ensure to remove the 30% sucrose/PBS solution around the sample.
  2. Dry the DRG sample by placing it on a dry Petri dish (Figure 2A) and moving it from one location to another two or three times with dry tweezers (Figure 2B).
  3. Dry the tweezers with lint-free tissue before moving another DRG sample (Figure 2C).
2. Put the dry DRG onto the base OCT.
  1. With dry tweezers, place the dry DRG onto the base OCT at the 12 o'clock position (Figure 1D).
  2. Keep at least 5 mm between the upper edge of the DRG and the upper edge of the base OCT.
  3. Put the dorsal part of the DRG at the 12 o'clock position and the ventral part at the six o'clock position (Figure 1D).
3. Embed the DRG tissue with OCT.
  1. Add more OCT to cover the entire DRG sample in a round or oval shape, with the DRG in the center (Figure 1E).
  2. Ensure the upper edge of the OCT covers the DRG at the upper edge of the base OCT (Figure 1F), as this makes it easier to transfer the section onto the glass slide (Figure 3A).
    NOTE: If the cover OCT is in the middle of the base OCT, the upper edge of the base OCT will block the glass slide to collect the section (Figure 3B).
  3. Freeze the cover OCT and DRG sample at -20 °C for another 5 min (Figure 1G). Do not trim the OCT around the DRG sample.
  4. Cut off the top of the cover OCT at a 30 µm thickness until the DRG is visible.

2. Cryosectioning of the DRG

Change the cryostat section thickness to 12 µm to cut DRG sections onto the slide (Figure 1H).
Hold the OCT section at the bottom with a small paintbrush cooled to -20 °C.
NOTE: It is important not to cut the whole section completely, but to leave 1-3 mm uncut (Figure 1I).
Use the end of a room-temperature tweezer to gently touch the bottom (six o'clock position) of the section so that it sticks to the platform surface (Figure 1J). This prevents the section from curving back in a roll, which makes it difficult to collect the section on the slide.
NOTE: When the bottom of the section sticks to the platform surface, and the top of the section still connects with the cover OCT, the section is in a flat shape (Figure 1K), making it much easier to collect the section on the slide.
Take a charged glass slide (see Table of Materials) and slowly place the slide over the section. As soon as the section starts to stick to the slide, gently pull the slide backward (Figure 1L).
Follow the same process to get more DRG sections onto the slide without overlapping the sections (Figure 1M). Ensure that a new DRG section is not placed over the OCT of another DRG section (Figure 4), as such a section cannot stay well on the slide and can be washed away during the immunochemistry staining process.

3. Nissl staining of the DRG section on the glass slide

Wash the sections for 10 min in PBS plus 0.1% Triton X-10. Then, wash the sections twice with PBS.
Apply a 1:300 Nissl stain (see Table of Materials) to the slide and incubate for 20 min at room temperature.
Wash the section with PBS plus 0.1% Triton X-100. Then, wash the sections twice with PBS for 5 min, and then for 2 h at room temperature.
Apply 4′,6-diamidino-2-phenylindole (DAPI) fluoromount-G (see Table of Materials) to cover the sections with a coverslip.

Representative Results

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 DRG sections on the glass slides. Figure 5 shows the confocal fluorescence microscopy image of the physiological structure of mouse DRG sections. Nissl staining shows the various-sized DRG sensory neurons, and DAPI staining shows the cells surrounding the neurons. This image demonstrates the quality of the DRG sections obtained with this protocol. Different antibodies can be applied for the immunohistochemistry studies of DRG, including studying different neuron types in the DRG.

Figure 1: Step-by-step procedure for cryosectioning mouse DRGs. (A) OCT is frozen on the top surface of the aluminum block. (B) The top of the OCT is cut off flat. (C) A mark is made at the bottom (six o'clock position) of the base OCT. (D) The DRG is put on the base OCT, with the ventral part facing the mark. (E) More OCT is added to cover the entire DRG sample. (F) The upper edge of the OCT covering the DRG needs to be at the upper edge of the base OCT. (G) The cover OCT and DRG sample is frozen. (H) The top of the cover OCT is cut off till the DRG tissue is visible (marked with the red arrow). (I) The OCT section is held at the bottom with a pre-cooled paintbrush. (J) The bottom of the section is stuck on the platform surface using the end of room-temperature tweezers. (K) Another view of (J) shows that the bottom of the section sticks to the platform surface and the top still connects with the cover OCT. (L) The section is stuck to a glass slide. (M) The same process is repeated to get more DRG sections onto the slide without overlapping sections. Please click here to view a larger version of this figure.

Figure 2: Technique to remove extra liquid from the DRG tissue. (A) The DRG tissue is placed on a dry Petri plate directly from a 30% sucrose solution, which has a lot of surrounding liquid. (B) The DRG tissue is moved to a neighboring location on the Petri plate using dry tweezers to remove the extra surrounding liquid. (C) The tweezers are dried with a lint-free wipe every time after moving the DRG tissue. Please click here to view a larger version of this figure.

Figure 3: The importance of properly placing the cover OCT on the base OCT. (A) The ideal place of the cover OCT: (A-1) The upper edge of the OCT covering DRG tissue should be at the upper edge of the base OCT. (A-2) The section should not be cut completely, with a small part connected to the upper edge of the cover OCT. (A-3) The section is transferred onto the glass slide smoothly. (A-4) The drawing shows that, in terms of the section at the upper edge, it is easy to transfer the section onto the glass slide. The black curved line indicates the section that has been cut. (B) Improper location of the cover OCT. (B-1) The OCT covering DRG tissue is placed in the center of the base OCT. (B-2) The section is not completely cut, with a small part connected to the upper edge of the cover OCT. (B-3) The upper edge of the aluminum block prevents the glass slide from touching the section. The red arrows indicate the distance between the glass slide and the cut section. (B-4) The drawing shows that the glass slide is blocked before touching the section. The black curved line indicates the section that has been cut. Please click here to view a larger version of this figure.

Figure 4: The proper placement of DRG sections on the glass slide. (A) The correct method to place the DRG sections next to each other without overlapping the DRG section and the OCT of other DRG sections. (B) The incorrect method to place the DRG sections on the OCT of another DRG section. Because the second DRG section does not directly stick onto the glass slide, it can be easily washed away during the subsequent processes. Please click here to view a larger version of this figure.

Figure 5: Nissl staining of mouse L4 DRG. An example of a 20x confocal image of a mouse L4 DRG section. Green indicates Nissl (neuron), and red indicates DAPI (nucleus). The scale bar represents 50 mm. Please click here to view a larger version of this figure.

Discussion

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 is important to make a mark at the bottom (six o'clock position) of the base OCT and to keep this mark at the six o'clock position throughout the process. Keeping a constant orientation helps to get more DRG sections, as otherwise the DRG cannot be cut completely from top to bottom, and some DRG samples will be left out and wasted. Third, the upper edges of both the base OCT and cover OCT must be at the aluminum block's upper edge. In this way, the upper edge of the aluminum block will not block the glass slide from taking the DRG sections. Fourth, it is important to avoid putting the DRG section within the OCT of neighboring DRG sections, because the second DRG section will not directly stick on the glass slide, and thus can be easily washed away in the subsequent process.

DRG cryosectioning is a very important technology in investigating DRGs in the mechanisms of pain and itch¹^,². However, due to the very small volume of mouse DRGs, collecting proper cryostat sections of mouse DRG samples is often challenging. One major problem is that the DRG-containing OCT sections tend to curve into a roll, making it difficult to put the sections onto the glass slides. Another problem is that, because the mouse DRG sample is very thin, it is difficult to obtain sufficient DRG sections from one DRG sample if the cryosectioning settings are not optimized. Here, we presented a step-by-step protocol for mouse DRG cryosectioning, making collecting high-quality DRG sections easy and practical. With this protocol, we are able to collect about 16 continuous, high-quality DRG sections from one mouse L4 DRG.

One limitation of this protocol is that it cannot provide direct 3D reconstruction as the tissue-clearing technique²⁶. Nevertheless, as we can obtain continuous, high-quality sections with this method, we can take images from these continuous sections to reconstruct 3D images.

Although the protocol for the cryosectioning of DRGs is developed from young adult mice aged 8-12 weeks, this protocol can be applied for cryosectioning human DRGs and DRGs from mice of younger or older ages. In fact, this protocol should be able to be applied for the cryosectioning of any volume samples.

Declarações

The authors have nothing to disclose.

Acknowledgements

None.

Materials

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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He, L., Zhao, W., Zhang, L., Ilango, M., Zhao, N., Yang, L., Guan, Z. A Procedure for Mouse Dorsal Root Ganglion Cryosectioning. J. Vis. Exp. (196), e65232, doi:10.3791/65232 (2023).