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

Abstract In this study, quantitative digital image analysis was utilized to measure the optical density of immunostains of involucrin at different depths in the epidermis to obtain reliable ordinal-scaled interpretations of the staining intensity. The distribution of involucrin within the epidermis was investigated in air-liquid interface and submerged skin organ cultures at different time-points. A greyscale calibration procedure 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-liquid 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 already observed in the basal epidermis. The results suggest that air-liquid interface conditions maintained physiological conditions better than submerged conditions which result in cultures that may have to increase their involucrin synthesis to improve the barrier function against the surrounding liquid during culture. Alternatively, changes in involucrin synthesis could reflect disturbed homeostasis. Concentrating measurements 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. Received: 29 June 1998 / Received after revision: 28 October / Accepted: 2 November 1998