THE INTRACELLULAR-DISTRIBUTION OF VINCULIN AND ALPHA(2)-INTEGRIN IN EPITHELIAL-CELLS AND CHONDROCYTES

The purpose of this study was to demonstrate the presence of vinculin and alpha(2) integrin in chondrocytes in situ and epithelial cells. We also determined that the appropriate fixation and extraction protocol s for immunohistochemistry and laser scanning confocal microscopy for an integral membrane protein and an actin-associated protein in cultur ed cells and whole tissue was different. Cultured epithelial cells, wh ole mount human cornea and avian cartilage were fixed and prepared usi ng a number of standard procedures used for indirect fluorescence immu nohistochemistry. The distribution of vinculin was cell-type and fixat ion-specific. Chondrocytes and cultured epithelial cells demonstrated vinculin in areas that appear to be associated with filamentous actin. Vinculin was associated with cell membranes in human cornea. The expr ession of alpha(2) integrin observed in chondrocytes fixed with methan ol, paraformaldehyde, or formaldehyde is consistent with its role in c ell-substrate interaction, but may also suggest a role in dividing and differentiating cells. The localization of alpha(2) integrin in human corneal epithelia supports its role as a cell-cell adhesion molecule. The cytoplasmic distribution of vinculin and alpha(2) integrin in tis sues fixed without detergent extraction suggests that the fixation ste p may be sufficient for antibody penetration and antigen extraction. T hese studies are the first report of vinculin and alpha(2) integrin in embryonic chondrocytes. In addition we have shown that confocal laser scanning microscopy combined with proper fixation and extraction prot ocols may optimize the localization of antigens in cultured and whole mount cells.The purpose of this study was to demonstrate the presence of vinculin and alpha, integrin in chondrocytes in situ and epithelial cells. We also determined that the appropriate fixation and extractio n protocols for immunohistochemistry and laser scanning confocal micro scopy for an integral membrane protein and an actin-associated protein in cultured cells and whole tissue was different. Cultured epithelial cells, whole mount human cornea and avian cartilage were fixed and pr epared using a number of standard procedures used for indirect fluores cence immunohistochemistry. The distribution of vinculin was cell-type and fixation-specific. Chondrocytes and cultured epithelial cells dem onstrated vinculin in areas that appear to be associated with filament ous actin. Vinculin was associated with cell membranes in human cornea . The expression of alpha, integrin observed in chondrocytes fixed wit h methanol, paraformaldehyde, or formaldehyde is consistent with its r ole in cell-substrate interaction, but may also suggest a role in divi ding and differentiating cells. The localization of alpha, integrin in human corneal epithelia supports its role as a cell-cell adhesion mol ecule. The cytoplasmic distribution of vinculin and alpha, integrin in tissues fixed without detergent extraction suggests that the fixation step may be sufficient for antibody penetration and antigen extractio n. These studies are the first report of vinculin and alpha, integrin in embryonic chondrocytes. In addition we have shown that confocal las er scanning microscopy combined with proper fixation and extraction pr otocols may optimize the localization of antigens in cultured and whol e mount cells.