Detection of multiple fibronectin isoforms in fetal fibroblast monolayer culture: a novel method for the qualitative and quantitative detection of multiple antigens

Analyzing the expression of multiple distinct antigens within a single mono layer culture involves cumbersome immunostaining techniques. We describe a simple and economical procedure for the detection and quantification of mul tiple antigens within a single monolayer. By generating an immunohistochemi cal grid which divides a monolayer in a standard tissue culture dish into 2 0 distinct areas, we were able to detect and quantify four individual fibro nectin (FN) isoforms within a single fibroblast monolayer culture. Quantifi cation of each isoform was performed using a modified enzyme-linked immunoa ssay. In addition, within the same monolayer, each FN isoform was detected using standard immunohistochemical detection with DAB visualization. Using this novel approach to immunohistochemical analysis we determined that with in the first 4 days of culture, the quantity of all FN isoforms increases f aster than the number of cells. However, upon reaching confluency, the quan tity of FN/cell drops dramatically. After reaching confluency, the amount o f FN/cell levels off and remains constant within the postconfluent monolaye r. Statistical analysis of the quantity of FN/cell indicates that a signifi cant reduction in the amount of FN/cell occurs in the 2 days prior to reach ing confluency. The distribution of all the FN isoforms, with the exception of B-FN, was found along the length of the cell body. In contrast, the dis tribution of B-FN was altered in postconfluent monolayers where it was dete cted only in distinct locations within the monolayer.