Myofibroblasts are resident cells of wound healing, contractures and fibroses; these tissues are often referred to as fibroproliferative. Whether myofibroblasts themselves proliferate is of interest. Since many in vitro cultures are heterogeneous, staining in situ is required to identify the myofibroblast. We have tested a newly available fluorescent staining kit using ethynyl deoxyuridine (EdU) and click chemistry to identify EdU incorporation into the replicated DNA of proliferative cells. The proliferation stain was combined with the definitive myofibroblast immunostain for alpha smooth muscle actin (α-sma). Fibroblasts were grown on coverslips and within attached collagen lattices. Cultures were pulsed with EdU 4 h prior to fixation. Different standard methods of fixation and permeabilization were used to test the effects of these variables on EdU and α-sma labeling. Images of the stained samples were quantified as the total percentage of proliferative cells, as well as the proportion of fibroblasts and myofibroblasts that were proliferating. Proliferative myofibroblasts were identified in both culture conditions and with all preparation methods tested. Proliferation within the fibroblast population was greater than within the myofibroblast population in both culture conditions. Fixation and permeabilization had little effect on EdU or α-sma labeling. This method of identifying proliferative myofibroblasts will be useful in future studies of myofibroblast proliferation within heterogeneous populations.

Traditionally, diseases of myofibroblasts have been termed proliferative diseases. Our evidence suggests stimulation of the myofibroblast phenotype decreases cell number, either through apoptosis (cell death) or proliferation inhibition. Proliferation has been difficult to study using bromodeoxyuridine (BrdU) since it requires harsh conditions to demonstrate the stain. A commercially available kit (Click‐It) using a modified nucleotide, 5‐Ethynyl‐2′‐deoxyuridine (EdU), has eliminated the need for harsh conditions. We tested the kit's ability to identify proliferative myofibroblasts using cells grown on coverslips or within collagen lattices. Cells were grown on coverslips for 24 to 48 hours, and in lattices for 5 days, to allow the myofibroblast phenotype to develop. Cells were then incubated in the presence of 10mM EdU for up to 4 hours, then fixed. The recommended staining procedure was modified so that the volume could be reduced tenfold. Both coverslips and lattices demonstrated EdU staining and alpha‐smooth muscle actin incorporated into stress fibers. Four cell populations were identified, including the proliferative myofibroblast. This staining technique with modifications will be a useful and inexpensive method for determining how proliferation plays a role during myofibroblast phenotype modulation.