Multiplex immunohistochemistry (mIHC) facilitates the identification of multiple cellular fractions within tissue sections. The Peter Mac’s Centre for Advanced Histology and Microscopy (CAHM) has optimized several antibody panels for use in 7-color mIHC. Visualisation of target antigens is accomplished through Tyramide Signal Amplification (TSA). The primary antibody and secondary Horseradish Peroxidase-conjugated antibody are removed after each labelling round by boiling the section, leaving the fluorescently conjugated Tyramide clustered around the antigen. Antibodies raised in the same species can be used due to the removal of antibodies after each round of IHC. Sections labeled by mIHC are imaged with a multispectral camera on the Perkin Elmer Vectra microscope, or with a confocal microscope. Using confocal microscopy >7 fluorophores can be spectrally unmixed and fluors with a long stokes shift, which is the separation between excitation and emission wavelengths, can be used. The Vectra is currently limited to 7 fluorophores with an emission range between 420 and 720nm but has some advantages over the Confocal microscope: (i) the ability to automatically scan 200 sections, and (ii) the Vectra’s powerful analytical software, Inform, which can be trained to automatically segment regions or cells of interest.
Two major bottlenecks in exist in mIHC, the time taken to manually label each section with 7 different antibodies and the time taken to perform the subsequent analysis. We are currently evaluating the Leica BOND RX immunostainer for automating mIHC. Using this equipment, a 7-color mIHC procedure is reduced from 2-3 working days to an overnight run. Thirty slides can be labeled with different antibody panels at the same time and reproducibility is excellent. In future, we aim to provide high throughput mIHC to both researchers and industry.