In situ hybridization of immunoglobulin light chain mRNA in paraffin sections using biotinylated or hapten-labelled oligonucleotide probes

Abstract

An in situ hybridization technique has been developed for the detection of immunoglobulin light chain mRNA in routine pathology specimens. The method detects kappa or lambda constant region sequences using a cocktail of synthetic oligonucleotide probes labelled with biotin or fluorescein 5-isothiocyanate (FITC) reporter molecules. The probes were labelled at flanking sites chemically by primary amine directed acylation and by 'homopolymer tailing' with terminal deoxynucleotidyl transferase using non-radioactive nucleotide analogues. The mRNA was unmasked in the formalin-fixed tissue sections by digestion with varying concentrations of proteinase K, and the hybrids were demonstrated using alkaline phosphatase with either a streptavidin/biotin based four-stage system or an anti-FITC antibody based detection system. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results confirm that the method is specific for kappa or lambda mRNA and show that specific mRNAs can be detected in routine formalin-fixed sections using non-radioactive techniques with retention of good morphology. The method reliably detects light chain mRNA in cells expressing secretory immunoglobulin. The protocol can also be applied to tissue rich in endogenous biotin by using hapten-labelled probes.