A new method for double immunolabelling with primary antibodies from identical species

There are several double immunolabelling methods but each has its drawbacks. More often than not, antibodies with the required specificities are available in only one species and their use normally produces false labels due to cross-reactivity. We describe a new and reliable technique for staining with primary antibodies from the same species, that can even be employed on tissues of the donor species. The protocol avoids cross-reactivities without loss in sensitivity, uses commercially available reagents and takes advantage of enzymatic detection, although it can be adapted for fluorescent labelling. Briefly, tissue is incubated with one primary antibody, followed by a peroxidase-coupled secondary antibody which is detected using amino ethyl carbazol to give a red reaction product. Meanwhile, the next primary antibody is coupled in vitro to a biotinylated secondary antibody and excess binding sites quenched with normal immune serum from the same species as the primary antibody. This complex is applied to tissue and detected by the avidin-biotin/alkaline phosphatase technique using naphthol-AS-MX-phosphate/Fast Blue BB to produce a blue label. In addition to extensive controls, the reliability and broad applicability of this method has been confirmed in (1) murine skin cryostat sections to co-visualize antigen-presenting cells (MHC class II-immunoreactive; "-ir') with either antigen detecting T lymphocytes (CD4-ir) or Langerhans cells (NLDC-145-ir) and (2) locust (Insecta) abdominal ganglion paraffin sections, where it is known that immunoreactivities for octopamine and a FMRFamide-related peptide are colocalized in only one, uniquely identifiable neuron.