Detection of immunoglobulin light chain mRNA by in situ hybridisation using biotinylated tyramine signal amplification

Abstract

A highly sensitive method for the light microscopic in situ hybridisation of immunoglobulin light chain mRNA in formalin fixed, paraffin wax embedded sections is reported. This method is based on signal amplification using horseradish peroxidase catalysed deposition of biotinylated tyramine at the sites of hybridisation. κ and λ light chain immunoglobulin mRNA in situ hybridisation was performed with fluorescein isothiocyanate conjugated oligonucleotide probe cocktails. The hybridisation signal was detected using a biotinylated tyramine signal amplification procedure with streptavidinbiotin-horseradish peroxidase complex as the final layer. Peroxidase was demonstrated using 3,3'-diaminobenzidine. The biotinylated tyramine signal amplification method resulted in the sensitive detection of immunonoglobulin light chain mRNA, with the whole procedure being completed in one day. Moreover, the use of peroxidase as the final reporter molecule also allowed haemamatoxylin to be used as counterstain, thereby permitting the evaluation of cellular morphology.