Fluorescent in situ hybridisation on tissue sections: a quantitative approach with confocal laser scanning microscopy

The use of fluorescent detection methods in association with digital micros copy technologies is an innovative approach for tissue localisation of mess enger RNA. The success of such methods relies on the tissue preservation, l ocal availability of the probe and on the existence of high resolution trid imensional analysis systems. Cryostatic sections, mild denaturation, short oligonucleotide probes (20mer) and confocal laser scanning microscopy allow the fullfillment of all these conditions avoiding photobleaching and tissu e autofluorescence. In this paper, we describe in detail a method for in si tu hybridisation set up with digoxigenin-coupled oligonucleotide complement ary to p-actin mRNA as a probe and an anti-hapten fluorescent antibody as s econd step for detecting specific hybridisation. Fluorescence was analysed by means of a confocal laser scanning microscope (CLSM) that provides image s with low out-of-focus blurring also with relatively low numerical apertur e (NA) objectives. We propose also an easy method to perform semi-quantitat ive thresholding analysis which allows to discriminate between background a nd specific signal.