IMMUNOHISTOCHEMICAL METHODS FOR SEMIQUANTITATIVE ANALYSIS OF COLLAGENCONTENT IN HUMAN PERIPHERAL-NERVE

Methods are described for the semiquantitative analysis of the connect ive tissue components of human peripheral nerve using light microscopy . General histological preservation was assessed using haematoxylin an d eosin staining and the distribution of collagen type IV was investig ated using immunohistochemistry. Several techniques were investigated to establish the one giving optimum structural preservation, immunobin ding and greatest contrast for image analysis. Frozen sections were un suitable for this tissue and paraffin wax sections were therefore used . Alcohol fixation was rejected due to poor preservation of the endone urium, although immunobinding was excellent. Ice-cold formalin fixatio n for 24 h was found to be adequate for structural preservation and an tibody binding, provided that a protease step was introduced. Trypsin was found to be superior to pepsin for-exposing collagen type IV epito pes. Of the detection systems investigated indirect immunofluorescence was not suitable due to considerable autofluorescence of the nerve. T he avidin-biotin method provided the greatest contrast, and was theref ore the detection method of choice for image analysis. The optimum tec hniques for image analysis were then used on control human sural nerve to ascertain the best comparative method for collagen type IV in the perineurium. A method of semiquantitative analysis is described which takes into account the fact that there is a close linear relationship between collagen content per unit of:perineurium and perineurial perim eter as fascicle size increases in peripheral nerve. This means that d ata from 2 different sample groups call easily be compared, provided t hat a range of fascicle sizes is analysed in each case.