Efficient, versatile, powerful

Traditional immunohistology studies involve labelling a few antibodies at a time across sequential tissue sections. The power, efficiency and ultimate insights of these studies can be substantially improved by using high-plex immunofluorescence labelling on a single tissue section.

Here, we present a high-content multiplex immunohistochemistry (IHC) approach that improves the throughput of current methods but maintains accessibility in set-up time and cost by using commercially available reagents, standard immunofluorescence labelling protocols and conventional widefield microscopy equipment with relatively low-cost modifications.

Based on the protocol by Maric et al, 2021 we use commercially available primary and secondary antibodies, taking advantage of different host species and immunoglobulin types/subtypes with appropriately matched fluorophore-conjugated secondary antibodies.

Each labelling round is imaged using a standard widefield epifluorescence microscope equipped with commercially available narrow bandpass excitation/emission filters to spectrally separate each fluorophore, with minimal spectral crosstalk.

Repeated cycles of antibody stripping and relabelling are followed by automated alignment of images using the DAPI channel (nuclei staining) from each labelling/imaging cycle for spatial registration at the pixel level. A custom python script has been developed for this process.

The multiplex IHC labelling approach is being established as a collaborative research platform at The Centre for Brain Research in the Faculty of Medical and Health Sciences, University of Auckland.