Imaging of adipose tissue

Abstract

Adipose tissue is an endocrine organ that specializes in lipid metabolism and is distributed throughout the body in distinct white adipose tissue (WAT) and brown adipose tissue (BAT) depots. These tissues have opposing roles in lipid metabolism with WAT storing excessive caloric intake in the form of lipid, and BAT burning lipid through nonshivering thermogenesis. As accumulation of lipid in mature adipocytes of WAT leads to obesity and increased risk of comorbidity (Pi-Sunyer et al., 1998), detailed understanding of the mechanisms of BAT activation and WAT accumulation could produce therapeutic strategies for combatting metabolic pathologies. As morphological changes accompany alterations in adipose function, imaging of adipose tissue is one of the most important tools for understanding how adipose tissue mass fluctuates in response to various physiological contexts. Therefore, this chapter details several methods of processing and imaging adipose tissue, including bright-field colorimetric imaging of paraffin-sectioned adipose tissue with a detailed protocol for automated adipocyte size analysis; fluorescent imaging of paraffin and frozen-sectioned adipose tissue; and confocal fluorescent microscopy of whole mounted adipose tissue. We have also provided many example images showing results produced using each protocol, as well as commentary on the strengths and limitations of each approach.

Keywords: Adipose; Cell profiler; Confocal; Frozen; Lineage tracing; Paraffin; Whole mount.

Figure 1

A depiction of a slide prepared for imaging of adipose tissue in whole mount.

Figure 2

Example images of whole mounted fluorochrome-containing WAT acquired through confocal microscopy. WAT was isolated from fluorescent reporter mice (eGFP and dTomato) and/or incubated in fluorescent stains/antibodies to label lipids (LipidTox), plasma membranes (Cell Mask Orange), endothelial cells (Isolectin GS-IB₄), macrophages (F4/80 and CD11b), non-specific blood lineage cells (CD45), B-cells and adipocyte progenitor cells (CD24) as described in Table 1. Images were acquired with the appropriate laser/filter settings listed in Table 1 and laser voltage/gain settings determined by FMO controls.

Figure 3

A depiction of paraffin embedding of adipose tissue.

Figure 4

Example Images and Graph of Cell Profiler Adipocyte Size Analysis. (A) Brightfield image of H&E stained paraffin sectioned WAT. (B) Fluorescent image of H&E stained paraffin sectioned WAT using the Texas Red filter cube. (C) Image from (B) outputted from Cell Profiler 2.0 following adipocyte pixel area analysis. Yellow arrows indicate improperly gated cells that must be manually excluded from the dataset. (D) Adipocyte cell size distribution in WAT depots isolated from standard- (SD) and high fat-diet (HFD) fed mice following Cell Profiler 2.0 analysis and conversion of measured adipocyte pixel area to adipocyte diameter as described in section 3.3.

Figure 5

Immunohistochemistry on Paraffin Sectioned WAT. (A) HRP-DAB mediated immunolabelling of PdgfRα in paraffin sectioned WAT. Black arrows indicate PdgfRα+ cells marked by deposition of oxidized DAB, which is brown in color. (B) HRP-TSA mediated immunolabelling of phospho-SMADs 1,5,8 in paraffin sectioned WAT. White arrows indicate phospho-SMAD+ cells marked by fluorescence of oxidized TSA-A594.

Figure 6

Immunofluorescent staining of frozen sectioned WAT. (A) A classic crown-like structure stained with antibodies for F4/80 and Ki67 and counterstained with DAPI. (B) Sympathetic innervation of a beige adipocyte as shown through staining with antibodies for UCP-1 and the sympathetic nerve fiber marker tyrosine hydroxylase (TH) and counterstained with DAPI. Scale bar is 20 µm.