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

In this study, quantitative digital image an- alysis was utilized to measure the optical density of im- munostains of involucrin at different depths in the epi- dermis to obtain reliable ordinal-scaled interpreta- tions of the staining intensity. The distribution of in- volucrin within the epidermis was investigated in air- liquid interface and submerged skin organ cultures at different time-points. A greyscale calibration proce- dure to standardize the optical units was used. By the 2nd day of culture, staining of involucrin had shifted markedly towards the mid or basal epidermis. Air-liq- uid interface cultures showed a less intensive shift than the submerged cultures. Up to the 7th day, involucrin staining remained in the upper epidermis in the air- liquid interface cultures, though weak staining was al- ready observed in the basal epidermis. The results sug- gest that air-liquid interface conditions maintained physiological conditions better than submerged condi- tions which result in cultures that may have to increase their involucrin synthesis to improve the barrier func- tion against the surrounding liquid during culture. Al- ternatively, changes in involucrin synthesis could re- flect disturbed homeostasis. Concentrating measure- ments on certain cell layers might give more detailed information about changes in involucrin expression. Although the detection method was used to study the histochemistry of skin, it could easily be applied to other tissues as well.