Localisation of connective tissue and inhibition of autofluorescence in the human optic nerve and nerve head using a modified picrosirius red technique and confocal microscopy

The use of picrosirius red to localise connective tissue in thin tissue sec tions viewed by bright-field microscopy is well documented. Its use on thin tissue sections imaged by fluorescence confocal microscopy has also been r eported. Here we describe modifications to published procedures that allow picrosirius red staining of thick 60-mu m sections and their subsequent ana lysis by confocal microscopy. The use of phosphomolybdic acid pre-treatment was found to be essential for confocal analysis; in addition to preventing non-specific staining, it also quenched tissue autofluorescence. By incuba ting sections free-floating, pre-treating them with phosphomolybdic acid fo r 30 min and imaging them using an argon ion laser we were able to use conf ocal microscopy to image the entire depth of 60-mu m human optic nerve and nerve head sections stained with picrosirius red. The application of this m odified picrosirius red and confocal microscopy technique should be useful for analysing the three-dimensional structure of the optic nerve and other tissues with a similarly complex arrangement of connective tissue. (C) 1999 Elsevier Science B.V. All rights reserved.