Methylene Blue Assay for Estimation of Regenerative Re-Epithelialization In Vivo

Abstract The rapidity with which epithelial cells cover a wound surface helps determine whether scarring or scar-less healing results. As methylene blue is a vital dye that is absorbed by damaged tissue but not undamaged epidermis, it can be used to assess wound closure. We sought to develop a quantitative methylene blue exclusion assay to estimate the timeframe for re-epithelialization in regenerating appendages in zebrafish and axolotls, two classic model systems of regeneration. Following application of methylene blue to the amputation plane and extensive washing, the regenerating tail was imaged in vivo until staining was no longer visible. The percent area of the amputation plane positive for methylene blue, representing the area of the amputation plane not yet re-epithelialized, was measured for each time point. The loss of methylene blue occurred rapidly, within ~2.5 h in larval and juvenile axolotls and <1 h in adult zebrafish, consistent with high rates of re-epithelialization in these models of regeneration. The assay allows simple, rapid estimation of the time course for regenerative re-epithelialization without affecting subsequent regenerative ability. This technique will permit comparison of re-epithelialization across different strains and stages, as well as under the influence of various pharmacological inhibitors that affect regeneration.