{"doi":"10.17504/protocols.io.rm7vzkm14vx1/v1","title":"812.2 Lung FFPE Multiplexed Immunofluorescence Phenocycler-Fusion® Antibody Validation Protocol v1","abstract":"This protocol describes validation of a 45-antibody panel used for multiplexed immunofluorescent (MxF) staining and imaging of FFPE lung tissue sections utilizing the Phenocycler-Fusion® 2.0 platform (Akoya Biosciences). The validation strategy involves staining two serial lung sections, one with a cocktail of 45 antibodies either custom (in-house) barcode-conjugated antibodies or antibody-barcoded conjugates sourced from Akoya Biosciences, and the second with the respective carrier-free, unconjugated (prior to conjugation with oligo barcode tag) control antibodies. The commercially conjugated Akoya-sourced antibodies were omitted from the second section. In addition, a section of kidney cortex was exposed to the fully conjugated antibody cocktail to confirm specificity of antibodies directed against lung specific markers, and specificity of pan-epithelial, endothelial, megakaryocyte, and immune cell markers in 2 different tissues (e.g. lung, kidney) as cross-organ, high level cell type validation. The protocol includes sections describing 1) Tissue sections, 2) Labeling with barcode-conjugated or unconjugated Ab, 3) Reporter Plate and Experimental Design, 4) Multiplexed Imaging and Image Upload to OMERO for review, and 5) Review criteria. Detailed descriptions of staining, imaging and conjugation procedures can be found in a companion protocol dx.doi.org/10.17504/protocols.io.6qpvr38dpvmk/v3.","journal":null,"year":2024,"id":492822,"datarank":0.10397207708399181,"base_score":0.6931471805599453,"endowment":0.6931471805599453,"self_citation_contribution":0.10397207708399181,"citation_network_contribution":0.0,"self_endowment_contribution":0.10397207708399181,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9324,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":12155,"name":"Gloria Pryhuber","orcid":"0000-0002-9185-3994","position":1,"is_corresponding":false},{"id":561600,"name":"Heidie Huyck","orcid":"0000-0001-9453-0747","position":2,"is_corresponding":false},{"id":1339539,"name":"gail.deutsch not provided","orcid":null,"position":3,"is_corresponding":false},{"id":1195605,"name":"Jeffrey M. Purkerson","orcid":"0000-0001-7636-4992","position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:08:56.210382Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}