A detailed protocol for open and low-cost six-plex immunofluorescence (Flex-6 mIHF) with a proof-of-concept study on...

Multiplex immunohistofluorescence (mIHF) allows to investigate protein (co)-localisation in the tumour microenvironment, which can facilitate research on carcinogenesis. Most available technologies for multiplex staining are expensive and immutable. We aim to demonstrate the feasibility and the flexibility of a low-cost mIHF protocol through the illustrated fine-tuning of two six-plex panels on human breast cancer samples. We detail a workflow combining two fluorescence amplification steps - (1) a peroxidase-labelled polymer conjugated to secondary antibodies and (2) tyramide signal amplification technology - to overcome the high autofluorescence of FFPE sections. The optimised slide scanner configuration enables single-run acquisition of up to six markers plus a nuclear dye from one tissue section. The resulting native scans can be directly used for analysis without the need for extensive or complex image processing. As a proof of concept, this protocol was applied on breast tissue samples from 19 patients diagnosed with ductal carcinoma in situ (DCIS), of various size, age and fat content. Two six-plex panels highlighted proteins expressed in tumours cells, extracellular matrix, and lymphocytes. The 12 proteins were first individually validated by immunohistochemistry, subsequently by immunohistofluorescence and finally combined in two six-plex panels. Only one sample could not be interpreted. Some samples displayed tissue detachment, cold zones or heterogeneous immunoreactivity, independently of the fat content, surgical procedure or specimen age. Here, we propose an open, flexible and cost-effective six-plex protocol (Flex-6 mIHF) and a validation workflow. We demonstrated its feasibility on challenging tissue containing fat such as breast cancer tissues.