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In This Article

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

Summary

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Abstract

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

Introduction

Adipose tissue is an essential organ for energy storage and is characterized by dynamic remodelling and nearly unlimited expansion1. In addition to energy homeostasis, adipose tissue also plays an essential role in hormone secretion of over 50 adipokines to modulate whole-body metabolic function2. Adipose tissue has a diverse architecture comprising of various cell types including mature adipocytes, fibroblasts, endothelial cells, immune cells, and adipocyte progenitor cells3. Recent studies have shown that obesity and other metabolic dysfunction can significantly alter adipose tissue function and....

Protocol

All experimental animal protocols were approved by the Animal Care Committee of The Center for Phenogenomics (TCP) conformed to the standards of the Canadian Council on Animal Care. Mice were maintained on 12-h light/dark cycles and provided with free access to water and food. 7 month old C57BL/6J male mice were used in the whole-mount staining experiment.

NOTE: Sections 1 to 2 are in chronological order, with section 3 being an optional step right after section 1. Section 4 can be performed t.......

Representative Results

Due to the fragility of adipose tissue, methods involving multiple processing steps and sectioning can lead to disfigurement of adipose tissue morphology3 (Figure 1A). However, whole-mount staining can preserve the morphology of adipocytes, ensuring accurate interpretation of results (Figure 1B).

Over-fixation of adipose tissue leads to fixative-induced autofluorescence. As shown in Figure .......

Discussion

Although conventional techniques such as histology and cryosection offer benefits for observing intracellular structure, whole-mount staining provides a different perspective in adipose tissue research, which enables 3D visualization of cellular architecture of minimally processed tissue.

In order to successfully perform whole-mount staining, the following suggestions should be taken into consideration. Different adipose tissue depots can yield various immunostaining results; thus, the type of.......

Acknowledgements

This work was funded by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada, Pilot and Feasibility Study Grant of Banting & Best Diabetes Centre (BBDC), the SickKids Start-up Fund to H-K.S., Medical Research Center Program (2015R1A5A2009124) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning to J-R.K.

....

Materials

NameCompanyCatalog NumberComments
LipidToxLife TechnologiesH34477
PECAM-1 primary antibodyMilliporeMAB1398Z(CH)
TH (tyrosine hydroxylase) primary antibodyMilliporeAB152, AB1542
DAPI stainBD Pharmingen564907
Nikon A1R confocal microscopeNikonConfocal microscope
Ultramicroscope ILaVision BioTecLight sheet image fluorescent microscope
Alexa Fluor secondary antibodiesJackson ImmunoResearchWavelengths 488, 594 and 647 used
Purified Rat Anti-Mouse CD16/CD32BioSciences553141
DichloromethaneSigma-Aldrich270997
Dibenzyl-etherSigma-Aldrich33630
MethanolFisher ChemicalA452-1
30% Hydrogen PeroxideBIO BASIC CANADA INCHC4060
Dimethyl sulfoxide (DMSO)Sigma-AldrichD2650
GlycineSigma-AldrichJ7126
HeparinSigma-AldrichH3393
Lectin kit I, fluorescein labeledVECTOR LABORATORIESFLK-2100
F4/80Bio-RadMCA497GA
VECTASHIELD Hard Set Mounting Medium with DAPIVECTOR LABORATORIESH-1500
Paraformaldehyde (PFA)
Phosphate Buffer Saline (PBS)
Triton-X
Tween
Animal serum (goat, donkey)

References

  1. Sung, H. K. Adipose vascular endothelial growth factor regulates metabolic homeostasis through angiogenesis. Cell Metabolism. 17, 61-72 (2013).
  2. Greenberg, A. S., Obin, M. S. Obesity and t....

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