Immunohistochemical methods for semiquantitative analysis of collagen content in human peripheral nerve

Abstract

Methods are described for the semiquantitative analysis of the connective tissue components of human peripheral nerve using light microscopy. General histological preservation was assessed using haematoxylin and eosin staining and the distribution of collagen type IV was investigated using immunohistochemistry. Several techniques were investigated to establish the one giving optimum structural preservation, immunobinding and greatest contrast for image analysis. Frozen sections were unsuitable for this tissue and paraffin wax sections were therefore used. Alcohol fixation was rejected due to poor preservation of the endoneurium, although immunobinding was excellent. Ice-cold formalin fixation for 24 h was found to be adequate for structural preservation and antibody binding, provided that a protease step was introduced. Trypsin was found to be superior to pepsin for exposing collagen type IV epitopes. Of the detection systems investigated indirect immunofluorescence was not suitable due to considerable autofluorescence of the nerve. The avidin-biotin method provided the greatest contrast, and was therefore the detection method of choice for image analysis. The optimum techniques 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 perimeter as fascicle size increases in peripheral nerve. This means that data from 2 different sample groups can easily be compared, provided that a range of fascicle sizes is analysed in each case.