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Most available technologies for multiplex staining are expensive and immutable. We aim to demonstrate the feasibility and the flexibility of a low\u2010cost mIHF protocol through the illustrated fine\u2010tuning of two six\u2010plex panels on human breast cancer samples. We detail a workflow combining two fluorescence amplification steps \u2013 (1) a peroxidase\u2010labelled polymer conjugated to secondary antibodies and (2) tyramide signal amplification technology \u2013 to overcome the high autofluorescence of FFPE sections. The optimised slide scanner configuration enables single\u2010run 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\u2010plex 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\u2010plex 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\u2010effective six\u2010plex protocol (Flex\u20106 mIHF) and a validation workflow. We demonstrated its feasibility on challenging tissue containing fat such as breast cancer tissues.<\/jats:p>","DOI":"10.1111\/jmi.70068","type":"journal-article","created":{"date-parts":[[2026,2,14]],"date-time":"2026-02-14T06:11:11Z","timestamp":1771049471000},"page":"171-189","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["A detailed protocol for open and low\u2010cost six\u2010plex immunofluorescence (Flex\u20106 mIHF) with a proof\u2010of\u2010concept study on breast cancer tissue"],"prefix":"10.1111","volume":"302","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-8252-5111","authenticated-orcid":false,"given":"Julia A. 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