Methylene Blue Assay for Estimation of Regenerative Re-EpithelializationIn 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.

Electro-surgical management of a traumatic fibroma with clinical assessment of re-epithelialization using methylene blue assay: A clinico-histopathological case report

“…This makes it a simple yet effective technique to track the course of re-epithelialization. [ 16 ] As seen in the present case, methylene blue dye uptake gradually dwindled over 1 week and an indicative of re-epithelialization.…”

Quantitative and Semi-quantitative Methods for Assessing the Degree of Methylene Blue Staining in Sentinel Lymph Nodes in Dogs

“…Protocols for methylene blue stain analysis is not yet distinguished for whole organ specimens, however, ImageJ is utilized to detect methylene blue in cellular microscopy (22) or tissue staining (23). ImageJ detects staining, such as those for methylene blue and a similar dye called toluidine blue in applications relating to cellular staining and assays (24)(25)(26)(27). Based on the ImageJ user guide, detecting blue hues is straightforward and stringent, specifically when colors are convoluted or observers are colorblind (28).…”