Table 3.

Techniques used by researchers to evaluate the presence and degree of adipose tissue fibrosis.

Technique	Testing Method	Results
Sampling/Biopsies		Human subcutaneous adipose tissue samples can be gathered from live patients during bariatric surgery or cosmetic procedure (panniculectomy, abdominoplasty, liposuction, etc.) [21,79]. Needle biopsies can also be used while patients are under local anesthesia [79,156].
Staining	Hematoxylin and Eosin	Adipocyte morphology can be determined by staining with hematoxylin and eosin [79].
	Picrosirius Red	Collagen can be detected through histological imaging by staining with Picrosirius red [20,21,160]. The images can be analyzed to determine the collagen and adipocyte area in tissue cross sections.
	Masson’s Trichrome	Using Masson’s trichrome stain allows for collagen, mucus, nuclei, cytoplasm, keratin, muscle fibers, and erythrocytes to be stained [82].
	Pimonidazole hydrochloride	Pimonidazole hydrochloride can be used to stain cells that are in a hypoxic environment [82].
Imaging	Polarized Light Microscopy	Polarized light microscopy can detect different collagen types. Under polarized light and stained with Sirius red, type I collagen fibers will appear orange to red, while type II collagen fibers will appear yellow to green [19,160].
	Confocal Microscopy	Though traditional histological approaches allow important information to be discerned, fully understanding collagen volume and dispersity can only be evaluated using 3D imaging approaches. Confocal microscopy can be used to discern the differences in adipocyte size and collagen amount between healthy and fibrotic tissues [20,81].
	Second Harmonic Generation (SHG) Microscopy	SHG microscopy can be used to image collagen fibers without staining. This allows for 3-dimensional imaging to evaluate collagen dispersion and structure [81,180,181,182]. SHG microscopy has been paired with coherent anti-Stokes Raman scattering (CARS) and 2-photon fluorescence (TPF) to image adipocytes and elastin [183].
	Scanning Electron Microscopy (SEM)	SEM can be used to visualize adipocytes and ECM fibers. Researchers have used SEM to image collagen fibers [28].
	Transmission Electron Microscopy (TEM)	TEM can be used to see the interstitial space, caveolae, vasculature, and adipocytes [28].
Mechanical Testing	Tensile Testing	Tensile testing has been investigated as a method of measuring adipose tissue fibrosis [20]. By securing sections of fresh tissue between clamps the peak force and tensile strength can be determined. Samples with higher degrees of fibrosis will exhibit higher peak forces and tensile strengths. Custom made mechanical testing instruments have also been made [183].
	Rheological Testing	The storage modulus (G’) can be used to measure stiffness on the macroscale [81] which is correlated with increased collagen content.
	Atomic Force Microscopy (AFM)	AFM can be used to quantify stiffness on the microscale [184]. However, AFM only measures the surface stiffness rather than the interior of the sample.
	Shearwave Dispersion Ultrasound Vibrometry (SDUV)	SDUV allows tissue elasticity and viscosity to be measured noninvasively using imaging techniques [185].
	Magnetic Resonance Elastography (MRE)	MRE is a magnetic resonance imaging (MRI) technique. MRE allows for mechanical properties, like stiffness, to be investigated noninvasively using imaging techniques [185].
Gene Expression	PCR, RT-PCR, qPCR, QRTPCR	Specific genes have been linked to adipose tissue fibrosis, such as TGFβ1, αSMA, COL1, and COL6. Biopsied adipose tissue samples can be analyzed through PCR [21,82,154,156].
Assays	Hydroxyproline	Assays can be used to measure the abundance of hydroxyproline, a signature amino acid for fibrillar collagens [82,141].
	Glycerol	The levels of lipolysis can be quantified using a glycerol assay [81]. Lipolysis is thought to be affected by adipose tissue fibrosis with some studies having conflicting results [7,8,9].
Cell Type Frequencies	Flow Cytometry	By staining the cells with specific antibodies flow cytometry can be used to sort cells or count the number of cells in a population. Researchers have used this to quantify the frequency of SVF populations (stem cells, mast cells, and macrophages) [21,58].