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* These authors contributed equally
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.
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.
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....
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.......
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 .......
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.......
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.
....Name | Company | Catalog Number | Comments |
LipidTox | Life Technologies | H34477 | |
PECAM-1 primary antibody | Millipore | MAB1398Z(CH) | |
TH (tyrosine hydroxylase) primary antibody | Millipore | AB152, AB1542 | |
DAPI stain | BD Pharmingen | 564907 | |
Nikon A1R confocal microscope | Nikon | Confocal microscope | |
Ultramicroscope I | LaVision BioTec | Light sheet image fluorescent microscope | |
Alexa Fluor secondary antibodies | Jackson ImmunoResearch | Wavelengths 488, 594 and 647 used | |
Purified Rat Anti-Mouse CD16/CD32 | BioSciences | 553141 | |
Dichloromethane | Sigma-Aldrich | 270997 | |
Dibenzyl-ether | Sigma-Aldrich | 33630 | |
Methanol | Fisher Chemical | A452-1 | |
30% Hydrogen Peroxide | BIO BASIC CANADA INC | HC4060 | |
Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D2650 | |
Glycine | Sigma-Aldrich | J7126 | |
Heparin | Sigma-Aldrich | H3393 | |
Lectin kit I, fluorescein labeled | VECTOR LABORATORIES | FLK-2100 | |
F4/80 | Bio-Rad | MCA497GA | |
VECTASHIELD Hard Set Mounting Medium with DAPI | VECTOR LABORATORIES | H-1500 | |
Paraformaldehyde (PFA) | |||
Phosphate Buffer Saline (PBS) | |||
Triton-X | |||
Tween | |||
Animal serum (goat, donkey) |
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