Dec 28, 2023
812.1 Lung FFPE OMAP 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
Protocol Citation: Jeffrey Purkerson, Gloria S Pryhuber, Heidie Huyck, gail.deutsch 2023. 812.1 Lung FFPE OMAP Multiplexed Immunofluorescence Phenocycler-Fusion® Antibody Validation Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3b9olk5/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, 2023
Last Modified: December 28, 2023
Protocol Integer ID: 89890
Keywords: Phenocycler-Fusion, validation, multiplexed immunofluorescence, lung, tissue sections, FFPE
Funders Acknowledgement:
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 34-antibody panel used for multiplexed immunofluorescent (MxF) staining and imaging of FFPE lung tissue sections utilizing the Phenocycler-Fusion® platform (Akoya Biosciences).
The validation strategy involves staining two serial lung sections, one with cocktail of custom barcode-conjugated antibodies plus antibody-barcoded conjugates sourced from Akoya Biosciences and the second with the respective unconjugated, carrier-free control antibodies and omitting the Akoya antibodies.
In addition, a tonsil section was exposed to the all-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, tonsil).
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 and imaging procedures can be found in a companion protocol dx.doi.org/10.17504/protocols.io.6qpvr38dpvmk/v2.
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 specifically 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, in this case, Reporters-only, tests 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 tonsil 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 (D016-RLL-11B2-6), a known diseased Lung section (D115-RLL-11A2-19) and a Tonsil Section (Tonsil-11-5) was labeled with a current panel (Table 1) of barcode antibodies as described in dx.doi.org/10.17504/protocols.io.6qpvr38dpvmk/v2. The other lung serial section (D016-RLL-11B2-5) was labeled with a panel of carrier free, unconjugated antibodies used for custom conjugations from step 18.1 in the above protocol and are 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 | |
| SFTPC-BX020 | SFTPC | 1:500 | Invitrogen | PA5-71842 | |
| ß-III-Tubulin-BX055 | ß-III-Tubulin | 1:400 | R&D Systems | MAB1195 | |
| SCGB1A1-BX043 | SCGB1A1 | 1:400 | R&D Systems | MAB4218 | |
| SCGB3A2-BX002 | SCGB3A2 | 1:400 | Abcam | ab240255 | |
| CXCL4-BX004 | CXCL4 | 1:200 | Peprotech | 500-P05 | |
| PROX1-BX050 | PROX1 | 1:200 | R&D Systems | AF2727 | |
| LYVE1-BX025 | LYVE1 | 1:100 | R&D Systems | AF2089 | |
| RAGE-BX028 | RAGE | 1:100 | Abcam | ab228861 | |
| CD298-BX005 | CD298 | 1:100 | Abcam | ab167390 | |
| MUC5AC-BX040 | MUC5AC | 1:100 | Abcam | ab212636 | |
| SCEL-BX052 | SCEL | 1:100 | Abcepta | AP11564C | |
| TP63-BX006 | TP63 | 1:100 | Abcam | ab214790 | |
| COL1A1-BX054 | COL1A1 | 1:100 | Abcam | ab88147 | |
| EDNRB-BX027 | EDNRB | 1:50 | R&D Systems | MAB4496 | |
| CD1c-BX016 | CD1c | 1:50 | Novus | ab156708 |
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, Thermoscience) equilibrated in PBS in accordance with the manufacturer's recommendations.
Table 2. Antibodies commercially conjugated (Akoya Biosciences).
| Ab-Barcode | Dilution | Vendor | Cat # | |
| PanCK-BX019 | 1:200 | Akoya | 4450020 | |
| COLIV-BX042 | 1:200 | Akoya | 4450122 | |
| Keratin5-BX101 | 1:200 | Akoya | 4450090 | |
| ECAD-BX014 | 1:200 | Akoya | 4250021 | |
| CD45-BX021 | 1:200 | Akoya | 4550121 | |
| Ki67-BX047 | 1:200 | Akoya | 4250019 | |
| CD68-BX015 | 1:200 | Akoya | 4550113 | |
| CD14-BX037* | 1:200 | Akoya | 4450047 | |
| CD163-BX069 | 1:200 | Akoya | 4250079 | |
| CD4-BX003 | 1:200 | Akoya | 4550112 | |
| CD8-BX026 | 1:200 | Akoya | 4250012 | |
| CD3e-BX045 | 1:200 | Akoya | 4550119 | |
| FOXP3-BX031 | 1:200 | Akoya | 4550071 | |
| CD20-BX007 | 1:200 | Akoya | 4450018 | |
| HLADR-BX033 | 1:200 | Akoya | 4550118 | |
| CD11c-BX024 | 1:200 | Akoya | 4550114 | |
| MPO-BX098 | 1:200 | Akoya | 4250083 | |
| CD31-BX001 | 1:200 | Akoya | 4450017 | |
| SMA-BX013 | 1:200 | Akoya | 4450049 |
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. *CD14-BX037 was omitted from the OMAP-34 validation as the Ab was temporarily unavailable. However, this Ab has been validated on FFPE tissue by others and in prior experiments.
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 (Akoya Biosciences) as described in dx.doi.org/10.17504/protocols.io.6qpvr38dpvmk/v2. The Reporter Plate design and exposures used for validation of the Lung FFPE OMAP-34 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 OMAP-34 Reporter Plate Design and exposures
Cycle #, reporters and exposure times
| ATTO550 | Exp (ms) | Cy5 | Exp (ms) | AF750 | Exp (ms) | |
| Ki67-RX047 | 125 | CD68-RX015 | 25 | PanCK-RX019 | 150 | |
| CD8-RX026 | 150 | CD4-RX003 | 75 | CD20-RX007 | 150 | |
| Col1A1-RX054 | 400 | CD3e-RX045 | 50 | CD31-RX001 | 150 | |
| SFTPC-RX020 | 100 | HLADR-RX033 | 50 | SMA-RX013 | 150 | |
| CD298-RX005 | 100 | CD45-RX021 | 75 | CXCL4-RX004 | 150 | |
| TPSAB1-RX041 | 10 | FOXP3-RX031 | 150 | LYVE1-RX025 | 400 | |
| CD163-RX069 | 250 | CD11c-RX024 | 125 | RAGE-RX028 | 200 | |
| MPO-RX098 | 25 | COlIV-RX042 | 150 | Keratin5-RX101 | 75 | |
| ß-III-Tub-RX055 | 250 | CD1c-RX016 | 300 | SCEL-RX052 | 400 | |
| ECAD-RX014 | 200 | TP63-RX006 | 150 | SCGB1A1-BX043 | 125 | |
| SCGB3A2-RX002 | 75 | ENDRB-RX027 | 250 | MUC5AC-RX040 | 150 | |
| PROX1-RX050 | 150 | None | 150 | None | 150 |
DAPI exposure 3 milliseconds (ms), CD14-RX037 omitted due unavailability of Ab-barcode.
Multiplexed Imaging and Upload to OMERO for review
Multiplexed Imaging and Upload to OMERO for review
Tonsil and Lung Sections were imaged as described in dx.doi.org/10.17504/protocols.io.6qpvr38dpvmk/v2.
OMAP-34 Validation consisted of the following slide runs.
1. Tonsil 11-5 (OMAP-34 Antibody Panel)
2. D016-RLL-11B2-5 (Unconjugated Ab panel)
3. D016-RLL-11B2-6 (OMAP-34 Antibody Panel)
4. D115-RLL-11A2-19 (OMAP-34)
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. OMAP34validation) 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. Gloria Pryhuber (Principal investigator), and Dr. Gail Deutsch (Board Certified Pathologist, Pulmonary specialist).
Lung and tonsil 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, vascular endothelium, immune cell clusters), colocalization with appropriate pan-markers (e.g. CD4 with CD3/CD45, CD11c with HLADR/CD45; SCEL with RAGE and pan-cytokeratin (pan-CK), 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, with the exception of EDNRB-BX027, fulfilled this criterion. As a result, EDNRB was omitted from the Lung OMAP-34 panel.
Slides labeled with unconjugated Ab are assessed for the contribution, if any, of nonspecific reporter binding to generate fluorescent signal.
Expected result
Unconjugated Ab, or barcode contribute little if any fluorescent signal associated with cellular and structural features in lung tissue sections. The D016-RLL-11B2-5 section was essentially devoid of feature specific staining (compare left and right panels in Figure 1) and thus fulfilled this criterion.
Figure 1. D016-RLL-11B2-5 (section 5, Left Panel) labeled with unconjugated antibodies (Table 1) and imaged with the full Reporter panel (Table 3). D016-RLL-11B2-6 (section 6, Right panel) was labeled with the Barcode-Ab conjugated panel (Tables 1 and 2) and imaged with the full Reporter panel (Table 3), as for section 5 (left panel). The indicated channels are active in both left and right panel images.
The tonsil section(s) was reviewed for any nonspecific interactions of respective Ab-barcode conjugates expected to uniquely target lung specific markers (e.g. RAGE, SFTPC, SCGB1A1, SCGB3A2, MUC5AC).
Expected result
Lung specific markers do not stain cellular or structural markers in tonsil (See Figure 2). Ab-barcode conjugates identify the same cellular and anatomical features when present in both lung and tonsil (e.g., CD45, panCK). All Ab-barcode conjugates, with the exception of EDNRB-BX027 fulfilled this criterion.
Figure 2. Ab-barcode conjugates targeting lung specific markers did not stain cellular features in tonsil (Left Panel). Pan markers for epithelial (pan-cytokeratin) and immune cells (CD45) identify tonsil cell populations as expected (Right panel). The latter was true also for immune cell markers including CD20, CD3e, CD4, CD8, CD68 (data not shown).
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).