QUANTIFICATION IN IMMUNOHISTOCHEMISTRY - THE MEASUREMENT OF THE RATIOS OF COLLAGEN TYPE-I AND TYPE-III

Quantitative techniques in immunohistochemistry are needed, but they a re rarely applied because of doubtful reproducibility. We have develop ed a method for the detection of collagen types I and III in situ. The method applied was a two-step immuno-alkaline phosphatase technique w ith visualization of the end-product with Fast Red. The staining inten sity was measured with a microdensitometer and the results expressed a s ratios. The method yielded results that were unaffected by variation s in tissue section thickness but which were proportionally related to time and antigen concentrations. Leiomyoma tissue, with a ratio of co llagen types I and III of approximately 1.0, was used to establish the appropriate dilutions of the antibodies, thus assuring identical opti cal densities. By having the leiomyoma tissue sections incubated toget her with the heart tissue specimens, leiomyoma tissue was also helpful in correcting deviations from the 1.0 ratio. Accurate measurements of collagen type I/III ratios in normal human heart specimens were obtai ned with the present quantitative immunohistochemical technique.