Multiplex Immunofluorescence for Human Pancreatic Ganglia v1

Purpose: This protocol is a multiplex immunofluorescence (MxIF) protocol adapted from t-CyCIF (dx.doi.org/10.17504/protocols.io.bjiukkew) and bleach & stain (Bady et al. 2023) protocols. Our protocol involves sequential incubations of primary antibodies using combinations of Opal reagents and fluorescence conjugated secondary antibodies for tyramine signal amplification (TSA) visualization following by standard immunofluorescence for primary antibodies not requiring TSA. Each cycle can consist of 4 or more primary antibodies depending on imaging equipment. Within and between staining cycles, antigen retrieval is performed to strip primary and secondary antibodies. Between staining cycles, a bleaching procedure is included to quench autofluorescence and deposited Opal TSA fluorescence. This protocol can be adapted to include a wide variety of primary antibodies with different combinations of Opal reagents and fluorescence conjugated secondary antibodies. Expected Outcome: By carefully selecting combinations of primary antibodies, secondary antibodies, and Opal reagents, one can visualize numerous markers in a single tissue section while avoiding cross-reactivity. As described by Bady et al., this allows for up to 15-20 antibodies to be applied to a single tissue section rather than the typical 4-7 antibodies that can be used in a single run. Scope: This protocol can serve as reference source for immunostaining human pancreas endocrine cells and intrinsic autonomic ganglia and is expected to also work well for formalin-fixed paraffin-embedded samples.