Feb 10, 2025
812.2 Lung FFPE Multiplexed Immunofluorescence Phenocycler-Fusion® Antibody Validation Protocol
- Jeffrey Purkerson1,2,
- Gloria S Pryhuber3,2,
- Heidie Huyck3,2,
- gail.deutsch4,5,2
- 1University of Rochester;
- 2URMC HuBMAP Lung TMC;
- 3University of Rochester Medical Center;
- 4Seattle Children's Hospital;
- 5University of Washington Department of Pathology
- URMC Pryhuber Lab
Protocol Citation: Jeffrey Purkerson, Gloria S Pryhuber, Heidie Huyck, gail.deutsch 2025. 812.2 Lung FFPE Multiplexed Immunofluorescence Phenocycler-Fusion® Antibody Validation Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzkm14vx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 25, 2024
Last Modified: February 10, 2025
Protocol Integer ID: 110926
Keywords: Phenocycler-Fusion, validation, multiplexed immunofluorescence, lung, tissue sections, FFPE, kidney, organ mapping antibody panel (OMAP)
Funders Acknowledgements:
National Heart, Lung, and Blood Institute
Grant ID: U01HL148860
NHLBI (LungMAP HTC) URMC
Grant ID: U01HL148861
NIH Human BioMolecular Atlas Program (HuBMAP)
Grant ID: U54HL165443
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.
Attachments
Guidelines
In order to validate both commercial and custom conjugated antibody specificity, control tissue sections were selected for the expected presence of specific tissue cells and structures, expected to be selectively marked by the panel of antibodies to be tested. In multiplexed Ab panels, inclusion of antibodies directed against general or pan-markers of epithelial, endothelial, and immune cells is recommended, as they will serve as positive controls for tissue section quality and effectiveness of the staining protocol when assessing the presence or absence of staining by tissue specific markers. No primary antibody tests, in this case, Reporters-only without conjugated-antibodies, are also recommended to assess non-specific background.
Materials
A complete list of materials and supply can be found in the companion protocol referenced within.
Before start
Review HubMAP guidelines for Ab validation in the attached SOP for OMAP construction
Tissue Sections
Tissue Sections
Lung serial sections, and a kidney section were prepared in FFPE as described in dx.doi.org/10.17504/protocols.io.kxygxejwdv8j/v2
Labeling of Tissue Sections with barcode conjugated Ab or unconjugated Ab
Labeling of Tissue Sections with barcode conjugated Ab or unconjugated Ab
One of two serial healthy Lung sections (D438-RLL-16B4-17), and a Kidney cortex section (K2300768-5-3) was labeled with a 45-marker panel (Tables 1 &2) of barcode antibodies as described in dx.doi.org/10.17504/protocols.io.6qpvr38dpvmk/v3 The other lung serial section (D438-RLL-16B4-16) was labeled, for non-specific staining control, with a panel of carrier free, unconjugated antibodies used for custom conjugations from step 18.1 in the above protocol and as listed in Table 1.
Table 1. Antibodies used for custom conjugations.
| Ab-Barcode | Unconjugated Ab | Dilution | Vendor | Cat # | |
| TPSAB1-BX041 | TPSAB1 | 1:1000 | Abcam | ab2378 | |
| SOX9-BX046 | SOX9 | 1:1000 | Cell Signaling | 74021SF | |
| SFTPC-BX020 | SFTPC | 1:500 | Invitrogen | PA5-71842 | |
| MUC5AC-BX040 | MUC5AC | 1:500 | Abcam | ab212636 | |
| FOXJ1-BX036 | FOXJ1 | 1:500 | Thermofischer | 14-9965-82 | |
| GRP-BX030 | GRP | 1:500 | LSBio | LS-C755064 | |
| TUBB3-BX055 | TUBB3 | 1:400 | R&D Systems | MAB1195 | |
| SCGB3A2-BX034 | SCGB3A2 | 1:400 | Abcam | ab240255 | |
| KRT8-BX022 | KRT8 | 1:400 | Biolegend | 904804 | |
| CD55-BX027 | CD55 | 1:200 | R&D Systems | AF2009 | |
| PF4-BX004 | PF4 | 1:200 | Peprotech | 500-P05 | |
| LYVE1-BX025 | LYVE1 | 1:200 | R&D Systems | AF2089 | |
| ATP1A1-BX005 | ATP1A1 | 1:200 | Abcam | ab167390 | |
| SFTPB-BX010 | SFTPB | 1:200 | ThermoFischer | PA5-42000 | |
| SCGB1A1-BX043 | SCGB1A1 | 1:100 | R&D Systems | MAB4218 | |
| AGER-BX028 | AGER | 1:100 | Abcam | ab228861 | |
| COL1A1-BX054 | COL1A1 | 1:100 | Abcam | ab88147 | |
| TP63-BX006 | TP63 | 1:75 | Abcam | ab214790 | |
| CD1c-BX016 | CD1c | 1:75 | Novus | ab156708 | |
| SCEL-BX052 | SCEL | 1:75 | Abcepta | AP11564C | |
| MUC5B-BX049 | MUC5B | 1:75 | Abcam | AB87376 | |
| PROX1-BX050 | PROX1 | 1:50 | Abcam | ab236026 |
For antibodies containing sodium azide (0.05-0.1%) or trehalose (5%), buffer exchange was performed utilizing Zeba™ Spin Desalting columns 7K MWCO (89890, 2ml, Thermo-Fisher Scientific) equilibrated in PBS in accordance with the manufacturer's recommendations prior to conjugation with nucleic acid tag (ref).
Table 2. Antibodies commercially conjugated (Akoya Biosciences).
| Ab-Barcode | Dilution | Vendor | Cat # | |
| CD31-BX001 | 1:200 | Akoya | 4450017 | |
| KRT14-BX002 | 1:200 | Akoya | 4450031 | |
| CD4-BX003 | 1:200 | Akoya | 4550112 | |
| CD20-BX007 | 1:200 | Akoya | 4450018 | |
| SMA-BX013 | 1:200 | Akoya | 4450049 | |
| ECAD-BX014 | 1:200 | Akoya | 4250021 | |
| CD68-BX015 | 1:200 | Akoya | 4550113 | |
| PanCK-BX019 | 1:200 | Akoya | 4450020 | |
| CD45-BX021 | 1:200 | Akoya | 4550121 | |
| CD11c-BX024 | 1:200 | Akoya | 4550114 | |
| CD8-BX026 | 1:200 | Akoya | 4250012 | |
| FOXP3-BX031 | 1:200 | Akoya | 4550071 | |
| HLADR-BX033 | 1:200 | Akoya | 4550118 | |
| CD14-BX037 | 1:200 | Akoya | 4450047 | |
| COLIV-BX042 | 1:200 | Akoya | 4450122 | |
| CD3e-BX045 | 1:200 | Akoya | 4550119 | |
| Ki67-BX047 | 1:200 | Akoya | 4250019 | |
| CD163-BX069 | 1:200 | Akoya | 4250079 | |
| CAV1-BX086 | 1:200 | Akoya | 4550084 | |
| MPO-BX098 | 1:200 | Akoya | 4250083 | |
| Keratin5-BX101 | 1:200 | Akoya | 4450090 | |
| SOX2-BX102 | 1:200 | Akoya | 4250075 | |
| PDPN-BX121 | 1:200 | Akoya | 4250094 |
Note that unconjugated forms of barcode-conjugated antibodies purchased from Akoya Biosciences are not included for validation. The majority of Akoya-sourced antibody clones have previously been validated for Akoya multiplexed immunofluorescence assessment of FFPE tissue.
The antibody dilutions correspond to working dilutions for the respective barcode-conjugated Ab.
Reporter Plate and Experiment Design
Reporter Plate and Experiment Design
Reporter plate design and Phenocycler-Fusion run protocols were developed using the PhenoCycler Experiment Designer Software 2.1.0 (Akoya Biosciences) as described in dx.doi.org/10.17504/protocols.io.6qpvr38dpvmk/v3. The Reporter Plate design and exposures used for validation of the Lung FFPE OMAP-45 Panel is shown in Table 3. Note that all 3 slides included in the validation were exposed to the same reporter panel.
Table 3. Lung FFPE 45 Marker Panel Plate Design and exposures
Cycle #, reporters and exposure times
| ATTO550 | Exp (ms) | Cy5 | Exp (ms) | AF750 | Exp (ms) | |
| Col1A1-RX054 | 150 | HLADR-RX033 | 50 | CD31-RX001 | 100 | |
| SFTPC-RX020 | 125 | CD45-RX021 | 50 | CXCL4-RX004 | 150 | |
| ATP1A1-RX005 | 125 | FOXP3-RX031 | 150 | LYVE1-RX025 | 200 | |
| TPSAB1-RX041 | 15 | CD11c-RX024 | 125 | AGER-RX028 | 200 | |
| CD163-RX069 | 150 | COLIV-RX042 | 150 | KRT5-RX101 | 50 | |
| MPO-RX098 | 35 | CD1c-RX016 | 150 | SCEL-RX052 | 250 | |
| TUBB3-RX055 | 125 | TP63-RX006 | 75 | SCGB1A1-BX043 | 75 | |
| CDH1-BX014 | 150 | FOXJ1-RX036 | 150 | MUC5AC-RX040 | 75 | |
| KRT14-RX002 | 100 | CD55-RX027 | 100 | SCGB3A2-RX002 | 100 | |
| PROX1-RX050 | 300 | GRP-BX030 | 150 | CAV1-BX086 | 25 | |
| PDPN-BX121 | 150 | SFTPB-BX010 | 50 | MUC5B-BX049 | 250 | |
| SOX2-BX102 | 50 | SOX9-BX046 | 50 | KRT8-BX022 | 50 | |
| Ki67-RX047 | 75 | CD68-RX015 | 10 | CD20-RX007 | 150 | |
| CD14-BX037 | 75 | CD4-RX003 | 75 | ACTA2-RX013 | 100 | |
| CD8-RX026 | 150 | CD3e-RX045 | 75 | PanCK-RX019 | 50 |
DAPI exposure 3 milliseconds (ms)
Multiplexed Imaging and Upload to OMERO for review
Multiplexed Imaging and Upload to OMERO for review
Kidney and Lung Sections were stained and imaged as described in dx.doi.org/10.17504/protocols.io.6qpvr38dpvmk/v3.
45 Marker Panel Validation consisted of the following slide runs.
| Source ID | Sample ID | Lab ID | Tissue | Panel | |
| TBD | TBD | K2300768-5-3 | Kidney Cortex | 45-marker | |
| NA | NA | D438-RLL-16B4-16 | Normal Lung | Unconjugated Ab | |
| HBM365.VQTL.758 | HBM958.DRDX.792 | D438-RLL-16B4-17 | Normal Lung | 45-marker | |
| TBD | TBD | D260-RLL-15C3-2 | Normal Lung | 45-marker | |
| TBD | TBD | D264-RLL-12B3-2 | Normal Lung | 45-marker | |
| TBD | TBD | D271-RLL-15B7-3 | Normal Lung | 45-marker | |
| TBD | TBD | D273-RLL-15B3-2 | Normal Lung | 45-marker | |
| TBD | TBD | D292-RLL-19B4-2 | Normal Lung | 45-marker | |
| TBD | TBD | D341-RLL-17C4-2 | Normal Lung | 45-marker | |
| TBD | TBD | D342-RLL-18A6-11 | Normal Lung | 45-marker | |
| TBD | TBD | D346-RLL-21C5-2 | Normal Lung | 45-marker | |
| NA | NA | D288-RLL-10B2-2 | Asthmatic Lung | 45-marker | |
| NA | NA | D294-RLL-7A2-3 | Asthmatic Lung | 45-marker | |
| NA | NA | D376-RLL-7A2-2 | Asthmatic Lung | 45-marker |
TBD= To be determined; NA= Not Applicable
The resulting .qptiff images are converted to ome.tiffs utilizing command line: C:\>bfconvert -no-sas -series 0 -compression LZW -pyramid-resolutions 5 -pyramid-scale 2 INPUT.qptiff OUTPUT_ome.tiff.
Utilizing Omero importer, ome.tiff files are uploaded to the URMC Omero-CODEX folder and the respective validation subfolder (i.e. OMAP45 validation) for review.
Review Criteria
Review Criteria
Validation of marker signals is reviewed by a 3-person panel that includes the protocol author (MxIF Research Scientist, Dr. Jeff Purkerson), Dr. Gloria Pryhuber (Principal investigator), and Dr. Gail Deutsch (Board Certified Pathologist, Pulmonary specialist).
Lung and kidney sections are reviewed for marker fluorescence signal localization to appropriate anatomical structures (e.g. airway, vasculature) and cell types within anatomical structures (e.g. alveolar epithelia, submucosal glands, vascular and lymphatic endothelium, immune cell clusters), colocalization with appropriate pan-markers (e.g. MUC5AC or/and MUC5B with PanCK; SCEL with RAGE, SFTPB with SFTPC etc.).
Expected result
All Barcode-Ab conjugates identify specific cellular and anatomical features in lung tissue sections. Note that all Ab barcode conjugates in Table 1 fulfilled this criterion.
Slides labeled with unconjugated Ab are assessed for the contribution, if any, of nonspecific reporter binding of fluorescent signals.
Expected result
Unconjugated Ab plus reporter barcodes contribute little if any fluorescent signal associated with cellular and structural features in lung tissue sections. The section was essentially devoid of feature specific staining (compare Upper and Lower panels in Figure 1) and thus fulfilled this criterion.
Figure 1. D438-RLL-16B4-16 (section 16, Top Panel) was
labeled with unconjugated antibodies (Table 1) and imaged with the full
Reporter panel (Table 3). D439-RLL-16B4-17 (section 17, Bottom panel) was
labeled with the Ab-Barcode-conjugated panel (Tables 1 and 2) and imaged with
the full Reporter panel (Table 3), as for section 16 (Top panel). The indicated
channels are active in both panel images.
The Kidney section(s) was reviewed for any nonspecific interactions of respective Ab-barcode conjugates expected to uniquely target lung specific markers (e.g. AGER, SFTPC, SCGB1A1, SCGB3A2, MUC5AC, MUC5B, SFTPB, FOXJ1, TP63).
Expected result
Lung specific markers do not stain cellular or structural markers in Kidney Cortex (See Figure 2, Top Panel). Ab-barcode conjugates in the 45-marker panel identify appropriate cellular and anatomical features in kidney cortex. (Figure 2, bottom panel). All Ab-barcode conjugates fulfilled these criteria.
Figure 2. K2300768-5-3, Section 3) was labeled with barcode
conjugated antibodies listed in Tables 1 and 2 and imaged with the full
Reporter panel (Table 3). Top Panel: Ab-barcode conjugates targeting
lung specific markers did not stain cellular features in Kidney Cortex. Bottom
Panel: Several Ab-barcode conjugates labeled specific cell types, such
macrophages (CD163), or structure features in kidney cortex such as vasculature
(CD31, SMA), and renal tubule epithelium (PanCK, ECAD).
Protocol references
1. Andrea J. Radtke, Ellen M. Quardokus, Diane C. Saunders. SOP: Construction of Organ Mapping
Antibody Panels for Multiplexed Antibody-Based Imaging of Human Tissues. V2.0.0 Approved by: Katy Börner, Neil Kelleher, Ronald N. Germain (11/30/2022) Supported by the HuBMAP Consortium. (See attached).
Acknowledgements
Histological technical services provided by Arianna Pitonzo.