Cryo-Sectioning of viral tracer-labeled macaque brains

Cryo-Sectioning of viral tracer-labeled macaque brains

Mingchao Yan¹ and Zheng Wang^2*
1Institute of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, China; 
²School of Psychological and Cognitive Sciences; Beijing Key Laboratory of Behavior and Mental Health; IDG/McGovern Institute for Brain Research; Peking-Tsinghua Center for Life Sciences, Peking University, China; 
^* For correspondence: zheng.wang@pku.edu.cn

Abstract

This protocol comprises the process of cryo-sectioning of viral tracer-labeled macaque brains, starting from tissue preparation to fluorescent imaging.

Materials and Reagents

1. 4% Paraformaldehyde (PFA)
2. Optimal cutting temperature (OCT) compound 
3. Phosphate buffered saline (PBS)
4. 0.9% NaCl
5. 15 and 30% sucrose in PBS
6. Cryoprotectant solution (containing 30% ethanediol, 30% sucrose in PBS solution)

Equipment

1.Cryostat microtome (Leica CM1950)
2.Wide field microscope (Olympus VS120)
3.Confocal laser microscope (Nikon TiE)

Procedure

1.Perfusion

1). Animals were deeply anesthetized and subsequently subjected to transcardial perfusion to achieve effective fixation of brain tissue.
2). Perfusion was initiated using physiological saline solution (0.9% NaCl, pH = 7.2) to clear blood from the circulatory system, and the process continued until the effluent ran clear.
3). Subsequently, perfusion was carried out using ice-cold 4% paraformaldehyde (PFA) in 0.01 M phosphate buffered saline to fix the tissues.

2.Brain Extraction and Post-Fixation

1). Following perfusion, brains were carefully extracted and immersed in 4% PFA for post-fixation.
2). The brains were post-fixed in 4% PFA for a duration of 72 hours at 4°C to ensure comprehensive tissue fixation.

3.Dehydration

The fixed brains were first cut into blocks, then equilibrated sequentially in 15 and 30% sucrose in PBS until it sank to the bottom of the container.

4.Tissue Embedding

Fixed brains were embedded in optimal cutting temperature (OCT) compound and swiftly frozen using the freezing shelf of a cryostat microtome (Leica CM1950) for subsequent cryo-sectioning.

5.Sectioning and Preservation

1). Cryo-sectioning was performed using a cryostat microtome (Leica CM1950) to obtain serial sections of 50 μm thickness.
2). To prevent damage from freezing and thawing, sections were preserved at -20。C in a cryoprotectant solution containing 30% ethanediol and 30% sucrose in PBS (pH = 7.2).

6.Mounting of the Brain Sections and Imaging

1). Thawed brain sections were gently washed in PBS to remove excess cryoprotectant solution.
2). The brain sections were then mounted onto microscope slides (52mm x 76mm).
3). Finally, the sections were coverslipped using a mounting medium and subjected to imaging using both a wide-field (Olympus VS120) and confocal laser microscope (Nikon TiE).

Recipes

Cryoprotectant solution (1L):
300 ml ethanediol
700 ml 30% sucrose in PBS solution

Acknowledgments

This protocol was adapted from and used in Yan et al. (2022)

References

Yan, M., Yu, W., Lv, Q., Lv, Q., Bo, T., Chen, X., Liu, Y., Zhan, Y., Yan, S., Shen, X., Yang, B., Hu, Q., Yu, J., Qiu, Z., Feng, Y., Zhang, X. Y., Wang, H., Xu, F., & Wang, Z. (2022). Mapping brain-wide excitatory projectome of primate prefrontal cortex at submicron resolution and comparison with diffusion tractography. eLife, 11, e72534. doi:10.7554/eLife.72534