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Analyzing the expression of multiple distinct antigens within a single monolayer culture involves cumbersome immunostaining techniques. We describe a simple and economical procedure for the detection and quantification of multiple antigens within a single monolayer. By generating an immunohistochemical grid which divides a monolayer in a standard tissue culture dish into 20 distinct areas, we were able to detect and quantify four individual fibronectin (FN) isoforms within a single fibroblast monolayer culture. Quantification of each isoform was performed using a modified enzyme-linked immunoassay. 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 within the first 4 days of culture, the quantity of all FN isoforms increases faster than the number of cells. However, upon reaching confluency, the quantity of FN/cell drops dramatically. After reaching confluency, the amount of FN/cell levels off and remains constant within the postconfluent monolayer. Statistical analysis of the quantity of FN/cell indicates that a significant reduction in the amount of FN/cell occurs in the 2 days prior to reaching 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 distribution of B-FN was altered in postconfluent monolayers where it was detected only in distinct locations within the monolayer.