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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

Abstract

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.

Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

Introduction

The prevalence of obesity and associated comorbidities has reached epidemic levels, causing a tremendous socioeconomic burden1,2. Various mouse models have been developed to better understand the biological processes responsible for obesity3,4. Because central mechanisms are important for the regulation of energy homeostasis in these animal models, neuroanatomical studies of mouse brains have become a necessary technique in this field. However, the quality, reliability, and reproducibility of brain histology techniques vary considerably among laborator....

Protocol

C57BL/6J mice of both sexes (8-16 weeks of age) were used in the present study. Care of all animals and all procedures were approved by Baylor College of Medicine's Institutional Animal Care and Use Committees.

1. Perfusion

NOTE: Steps 1.1 - 1.6 are performed in a fume hood.

  1. Anesthesia
    1. Pour 5 mL of isoflurane (see the Table of Materials) onto a paper towel placed at the bottom of a desiccator. Introduce t.......

Representative Results

The flow chart of this protocol is briefly illustrated in Figure 1. This laboratory's cryosectioning procedure is demonstrated in Figure 2A, in which 5 brain samples were sectioned simultaneously. The mounting of brain sections is shown in Figure 2B, and a fully mounted slide with brain sections is illustrated in Figure 2C. In Figure 3, representative fluorescence immun.......

Discussion

This protocol provides an established method for neuroanatomical studies of the mouse brain, including perfusion, tissue sectioning, free-floating immunostaining, tissue mounting, and imaging. However, a few key details essential for consistent and reliable results must be optimized.

The quality of perfusion is critical for successful staining. Staining results might be affected if blood remains in the brain, given that blood cells (e.g., red blood cells) can generate an artificial 'positi.......

Acknowledgements

The investigators were supported by grants from the NIH (K01DK119471 to CW; P01DK113954, R01DK115761, R01DK117281, R01DK125480, and R01DK120858 to YX), USDA/CRIS (51000-064-01S to YX), and American Heart Association Postdoctoral Fellowship (#829565) to LT.

....

Materials

NameCompanyCatalog NumberComments
Alexa Flour 594 donkey anti-rabbit IgG (H+L)InvitrogenA21207
 30% SucroseVWR47030230 g Sucrose dissoved into 100 mL of PBS
 Neutral Buffered FormalinVWR16004-12810%, 25 °C, pH 6.8-7.2
1 mL Sub-Q SyringeBD309597
48 Well Cell Culture PlateCorning3548
6 Well Cell Culture PlateCorning3516
Antifading mounting media with DAPIVector LaboratoriesH-1200
Autoclavable plastic desiccatorThermo Scientific Nalgene5315-0150
AVP antibodyPhoenix PharmaceuticalsH-065-07
Cell StrainerCorning431752
Cryoprotectant bufferUser preferenceNot applicable20% glycerol, 30% ethylene glycol, and 50% PBS
IsofluraneCovetrus11695-6777-2
Leica DFC310FX microscopeLeicaNot applicable
Microscope Slide Boxes (50-place)VWRNot applicable
PBTUser preferenceNot applicable2.5 mL of Triton X-100 dissolved into 1000 mL of PBS
Perfusion two automated Perfusion SystemLeica39471005
Phosphate-buffered saline (PBS) 20xVWRVWRVE703-1L25 °C, pH 7.3-7.5, 1x composition:137 mM NaCl, 2.7 mM KCl, 9.8 mM Phosphate buffer
Slideing Microtome Microm HM450ThermoFisherMicrom HM450
Sodium ChlorideRICCA Chemical7220-320.9%, 25 °C, pH 7.4
Streptavidin Protein, DyLight 488ThermoFisher#21832
Triton X-100Sigma-Aldrich089k01921
WFA antibodySigma-AldrichL1516
Zeiss Axio Z1 ScannerZeissNot applicable
Zen 3.1 softwarescanner software

References

  1. Must, A., et al. The Disease burden associated with overweight and obesity. Journal of the American Medical Association. 282 (16), 1523-1529 (1999).
  2. Apovian, C. M. Obesity: definition, comorbidities, causes, and burden.

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