A Clearing Technique to Enhance Endogenous Fluorophores in Skin and Soft Tissue

Abstract

Fluorescent proteins are used extensively in transgenic animal models to label and study specific cell and tissue types. Expression of these proteins can be imaged and analyzed using fluorescent and confocal microscopy. Conventional confocal microscopes cannot penetrate through tissue more than 4-6 μm thick. Tissue clearing procedures overcome this challenge by rendering thick specimens into translucent tissue. However, most tissue clearing techniques do not satisfactorily preserve expression of endogenous fluorophores. Using simple adjustments to the BABB (Benzoic Acid Benzyl Benzoate) clearing methodology, preservation of fluorophore expression can be maintained. Modified BABB tissue clearing is a reliable technique to clear skin and soft tissue specimens for the study of dermal biology, wound healing and fibrotic pathologies.

Figure 1

(A) Schematic of modified BABB tissue clearing protocol. 1. Mouse tissue harvest and fixation, 2. Tissue dehydration in progressive concentrations of tert-butanol titrated to pH = 9.5, 3. Clearing in BABB reagent (1:2 ratio of tert-butanol to BABB titrated to pH 9.5). (B) Schematic (top two panels – top most shows application of Vaseline® surrounding the specimen on a glass slide, panel below shows suspension of the specimen in BABB reagent and application of cover slip) with example images complementing the top two panels (bottom two panels) of whole-mounted cleared tissue specimens. Specimen edge outlined with white dotted lines. (C) Cleared (right panel) versus non-cleared (left panel) intact mouse skin whole-mounted in BABB reagent (right panel) versus standard mounting reagent (left panel). (D) Cleared (right panel) versus non-cleared (left panel) healed wounds from the dorsal surface of a mouse (harvested at post-operative day 14) whole-mounted in BABB reagent (right panel) versus standard mounting reagent (left panel). Wound scar edges outlined with black dotted lines. (E) Examples of cleared (middle panel) versus non-cleared (left panel) wound scar samples (harvested at post-operative day 14) demonstrating tissue shrinkage with modified BABB clearing. Graph (right panel) quantifies shrinkage of a set of specimens (n.s. = not significant, p > 0.01, paired t-test). Edge of non-cleared sample outlined with red dotted line and then overlaid on cleared specimen. Wound scar edges outlined with black dotted lines.

Figure 2

(A) Upper panels represent a z-stack image of cleared, uninjured L2G mouse skin (begins at left with deepest/dermal aspect, extending to most superficial/epidermal aspect at far right). Lower panels represent a z-stack image of non-cleared, uninjured L2G mouse skin from an adjacent area on the same animal as above. (Imaged on SP8 confocal microscope using the 20X objective, working distance = 680 μm with oil immersion, NA = 0.75). (B) Comparison of fluorescence intensity seen in cleared tissue from uninjured L2G mouse skin with comparable non-cleared tissue (*statistically significant, p < 0.0001, unpaired t-test). (C) Epidermal and dermal biology becomes distinctly visible with clearing. (White dotted lines highlight hair follicles, dermal fibroblasts indicated with yellow arrowheads, imaged on SP8 confocal microscope using the 40X objective, working distance = 240 μm with oil immersion, NA = 1.30). (D) 40X confocal imaging of cleared, uninjured L2G mouse skin whole-mounted with the hypodermis facing upwards (closest to the objective), shows that epidermis (farthest from the objective) can be clearly visualized through the full thickness of the tissue (red arrowheads highlight edges of keratinocyte “sheets” characteristic of epidermal biology, 40X objective, working distance = 240 μm with oil immersion, NA = 1.30). Experiments conducted with n = 3 biological replicates per condition (where applicable), scale bars represent 200 μm (unless otherwise indicated).

Figure 3

(A) Example of cleared, uninjured dermis from an αSMA-Cre^ERT2::Rosa26^mTmG mouse. Vasculature indicated by white arrowheads. (B) Example of cleared, uninjured dermis from a PDGFRα-Cre^ERT2::Rosa26^mTmG mouse. Blue arrowheads mark dermal fibroblasts. (C) Example of cleared, dermal scar tissue from an αSMA-Cre^ERT2::Rosa26^mTmG mouse. Blue arrowheads indicate activated dermal fibroblasts, neovascularization associated with wound healing indicated by white arrowheads, white asterisk marks the scar center, white dotted line highlights the closing edge of the scar. (D) Example of cleared, dermal scar tissue from a PDGFRα-Cre^ERT2::Rosa26^mTmG mouse. Blue arrowheads indicate dermal fibroblasts, white asterisk marks the scar center, white dotted line highlights the closing edge of the scar. Images taken using the 20X objective, working distance = 680 μm with oil immersion, NA = 0.75 on an SP8 confocal microscope (unless otherwise indicated). Experiments conducted with n = 3 biological replicates per condition (where applicable), scale bars represent 200 μm (unless otherwise indicated).

Figure 4

(A) Polyclonal expansion of keratinocytes involved in wound coverage (outlined with white dotted line), hair follicles near the edge of the wound show polyclonal proliferation (examples outlined with blue dotted lines), fibroblasts involved in wound healing (example outlined in yellow dotted line), in a cleared dorsal wound tissue section from an Actin-Cre^ERT2::Rosa26^VT2/GT3 mouse harvested at post-operative day 14. Merged images at left, individual channels at right (mCe = membrane (m) Cerulean, mG = mGFP, mOr = mOrange, mCh = mCherry). Images (A,B) taken at 20X using an SP8 confocal microscope (working distance = 680 μm with oil immersion, NA = 0.75), scale bars represent 200 μm. (B) Non-cleared tissue section equivalent to 4 A. (C) Cleared En1-Cre::Rosa26^mTmG peri-wound tissue. White arrowheads indicate En1-lineage dermal fibroblasts. Images (C,D) taken using a 20X objective using standard fluorescence microscopy (Leica DMI4000 B automated inverse fluorescence microscope), scale bars represent 150 μm. (D) Non-cleared En1-Cre::Rosa26^mTmG peri-wound tissue equivalent to 4 C. Experiments conducted with n = 3 biological replicates per condition (where applicable), scale bars represent 200μm (unless otherwise indicated).