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 protocols for immunohistochemistry and laser scanning confocal microscopy 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 prepared using a number of standard procedures used for indirect fluorescence immunohistochemistry. The distribution of vinculin was cell-type and fixation-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 expression of alpha 2 integrin observed in chondrocytes fixed with methanol, paraformaldehyde, or formaldehyde is consistent with its role in cell-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 tissues fixed without detergent extraction suggests that the fixation step may be sufficient for antibody penetration and antigen extraction. These 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 protocols may optimize the localization of antigens in cultured and whole mount cells.
Hirsch
M S
MS
Department of Anatomy and Neurobiology, Boston University School of Medicine, MA 02118.
Law
L Y
LY
Trinkaus-Randall
V
V
Svoboda
K K
KK
D000818:Animals; D002356:Cartilage / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D002642:Chick Embryo; D003315:Cornea / Q000378:metabolism* / Q000648:ultrastructure; D004848:Epithelium / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006801:Humans; D039421:Integrin alpha2; D016023:Integrins / Q000032:analysis*; D018613:Microscopy, Confocal; D016707:Tissue Fixation; D016596:Vinculin / Q000032:analysis*
D039421:Integrin alpha2; D016023:Integrins; D016596:Vinculin