Visualization and analysis of whole depot adipose tissue neural innervation

Abstract

Summary Little is known about the diversity and function of adipose tissue nerves, due in part to the inability to effectively visualize the tissue’s diverse nerve subtypes and the patterns of innervation across an intact depot. The tools to image and quantify adipose tissue innervation are currently limited. Here, we present a method of tissue processing that decreases tissue thickness in the z-axis while leaving cells intact for subsequent immunostaining. This was combined with autofluorescence quenching techniques to permit intact whole tissues to be mounted on slides and imaged by confocal microscopy, with a complementary means to perform whole tissue neurite density quantification after capture of tiled z-stack images. Additionally, we demonstrate how to visualize nerve terminals (the neuro-adipose nexus) in intact blocks of adipose tissue without z-depth reduction. We have included examples of data demonstrating nerve subtypes, neurovascular interactions, label-free imaging of collagen, and nerve bundle digital cross-sections.

A clearing-free protocol for imaging intact whole adipose tissue innervation in mice

“…Only the most superficial structures will be visible. See Willows et al. (2021) for a comparison of both appropaches.…”

“… Autofluorescence Quenching. There are four approaches (used separately) that can be taken to mitigate tissue autofluorescence, each with their own pros and cons summarized here and in greater detail in Willows et al. (2021) .…”

“…Below we have provided examples of images captured using this protocol ( Figure 5 ). For additional examples, see previous publications using this protocol ( Blaszkiewicz et al., 2019 , 2020 ; Willows et al., 2021 ).

Figure 5 Imaging whole mount scWAT (A–H) A series of images captured of whole mount scWAT display various combinations of the immunostaining and imaging described in this protocol.

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“…This protocol has been optimized and validated for adipose neurovascular labeling. For complete details on the use and execution of this protocol, please refer to Willows et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Willows et al. (2021) .…”