Quantitative digital image analysis applied to demonstrate the stratified distribution of involucrin in organ cultured human skin

In this study, quantitative digital image analysis was utilized to measure the optical density of immunostains of involucrin at different depths in th e epidermis to obtain reliable ordinal-scaled interpretations of the staini ng intensity, The distribution of involucrin within the epidermis was inves tigated in air-liquid interface and submerged skin organ cultures at differ ent time-points. A greyscale calibration procedure to standardize the optic al units was used. By the 2nd day of culture, staining of involucrin had sh ifted markedly towards the mid or basal epidermis, Air-liquid interface cul tures showed a less intensive shift than the submerged cultures. Up to the 7th day involucrin staining remained in the upper epidermis in the air-liqu id interface cultures, though weak staining was already observed in the bas al epidermis, The results suggest that air-liquid interface conditions main tained physiological conditions better than submerged conditions which resu lt in cultures that may have to increase their involucrin synthesis to impr ove the barrier function against the surrounding liquid during culture. Alt ernatively, changes in involucrin synthesis could reflect disturbed homeost asis, Concentrating measurements on certain cell layers might give more det ailed information about changes in involucrin expression, Although the dete ction method was used to study the histochemistry of skin, it could easily be applied to other tissues as well.