TITLE: 813.1 Multiplexed Immunofluorescence Phenocycler-Fusion®  Imaging of FFPE Lung Sections
AUTHORS: Jeffrey Purkerson, Gloria S Pryhuber, Luis Colon, Heidie Huyck

[DESCRIPTION]
This protocol describes multiplexed immunofluorescent (MxF) staining and imaging of FFPE lung tissue sections utilizing the Phenocycler-Fusion platform (Akoya Biosciences).  The approach is based on the CODEX multiplexed immunofluorescence staining technology developed by Garry Nolan and colleagues (1). The protocol is closely aligned with the attached Phenocycler-Fusion User Guide provided by Akoya (2). 
The protocol includes A) FFPE lung tissue section preparation, B) Labeling of lung tissue sections with antibody-barcode conjugates, C) Barcode-Reporter plate and experiment design, D) Multiplexed imaging and analysis, E) Reference for H&E stain to follow MxF and F) Reference to custom antibody conjugation. Thus, the protocol is designed to provide information regarding specific reagents (i.e., antibodies), conditions (i.e., dilutions), and procedures used in multiplexed immunofluorescent staining of lung tissue.  

[BEFORE_START]
The Phenocycler Fusion system must be setup and calibrated by an installation engineer.  Akoya Biosciences will provide a list of materials required for training.

[GUIDELINES]
An overview of Multiplexed Imaging using barcode conjugated antibodies can be found in the attached Phenocycler-Fusion User Guide.  Onsite training in staining procedures, experimental setup and design, as well as image acquisition is provided by Field Application Scientists from Akoya Biosciences.



[STEPS]
SECTION: Lung section  preparation
1. Bake Sections to promote tissue adherence to the slide.
Since stained slides can be stored in storage buffer for a maximum 5 days without diminution of staining signal intensity, and Phenocycler-Fusion imaging typically ranges from 16-24h, staining more than 5 slides at a time is not recommended.
SECTION: Lung section  preparation
1.2. Heat Slide(s) in oven at 60 °C   .
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
3. Rehydration
The tissue sections are rehydrated through descending ethanol series followed by molecular biology grade distilled water (2X).

100% Ethanol #1 5 min 
100% Ethanol #2 5 min 
90% Ethanol 5 min 
70% Ethanol 5 min 
50% Ethanol 5 min 
30% Ethanol 5 min 
ddH20 #1 5 min 
ddH20 #1 5 min 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
4. High pH Antigen RetrievalpH 9 
Heat-Induced Epitope Retrieval (HIER) reverses protein crosslinking in FFPE tissue
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
4.1. Dilute AR9 buffer (Akoya Biosciences) 1/10 in ddH20 and fill plastic Coplin Jar to 90-95% volume. Add slides containing tissue sections to be stained. Cover entire Coplin Jar with aluminum foil. (Do not Cap)

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
4.2. Place Coplin Jar in an InstantPot® pressure cooker containing ddH20 to 1/3-1/2 depth of Coplin Jar.
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
4.3. Heat on high pressure setting 20 min 

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
4.4. After releasing pressure per pressure cooker directions, remove Coplin Jar, partially unwrap foil without uncovering slides and allow slides to cool for a minimum of  60 min .  Attempting to rinse/wash without allowing slides to cool may reduce tissue adherence to glass slide.

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
5. Prepare Blocking Buffer 
Prepare Blocking Buffer no earlier than 1 h before staining (i.e. while slide are cooling in AR9 buffer and/or incubating in Staining buffer, see below) and keep on ice.
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
5.1. Table 1. Blocking Buffer Component Table 

Component            2 Slides            5 slides                  N Slides

Staining Buffer   362 µL  905 µL       181 µL 
N Blocker           9.5 µL    23.75 µL    4.75 µL 
G Blocker           9.5 µL    23.75 µL    4.75 µL 
J Blocker            9.5 µL    23.75 µL    4.75 µL 
S Blocker            9.5 µL    23.75 µL   4.75 µL 
Total Volume      400 µL   1000 µL   200 µL 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
5.2. Prepare Antibody Cocktail
In a 1.5 ml microfuge tube, mix volume of blocking buffer = 200 µL  minus total volume of antibodies (µl/slide x N) to be used in experiment (See Table below). The Blocking buffer volume must be ≥ 60% of total antibody cocktail volume for effective blocking.  If needed, reduce staining buffer volume to achieve 60% blocking buffer volume in Ab solution.

 Example antibody panel (final column = microliter of antibody per single slide):
     SFTPC-BX020 (1:500)      0.8      2
          TPSAB1-BX041
  (1:1000)      0.4      1
          CXCL4 (1:200)      2      5
          PanCK-BX019 (1:200)      2      5
          CD45-BX021 (1:200)      2      5
          CD68-BX015 (1:200)      2      5         ColIV-BX042(1:200)      2      5
          Ki67-BX047 (1:200)      2      5
          CD14-BX037 (1:200)      2      5
          CD4-BX003 (1:200)      2      5
          CD20-BX007 (1:200)      2      5         CD8-BX026 (1:200)      2      5
          CD3e-BX045 (1:200)      2      5
          CD31-BX001 (1:200)      2      5
          HLADR-BX033 (1:200)      2      5
          SMA-BX013 (1:200)      2      5
          FOXP3-BX031 (1:200)      2      5
          CD163-BX069 (1:200)      2      5
          Keratin5-BX101
  (1:200)      2      5
          LYVE1-BX025 (1:100)      4      10
          RAGE-BX028 (1:100)      4      10
          COL1A1-BX054
  (1:100)      4      10
          CD298-BX005 (1:100)      4      10
          CD11c-BX024 (1:200)      2      5
          MPO-BX098 (1:200)      2      5
          CD1c-BX016 (1:50)      8      20
          SCEL-BX052
  (1:100)      4      10
          β-III-Tub-BX055
  (1:400)      1      2.5
          ENDRB-BX027
  (1:40)      10      25
          SCGB1A1-BX043
  (1:400)      1
        2.5
          Antibody
  Buffer      332.8
  µL      832
  µL        TOTAL      400
  µL      1000
  µL        #Slides      2      5
      

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
6.2. Immerse Slides in sequential Coplin jars containing the following buffers from Akoya Biosciences:
Hydration buffer #1   2 min
Hydration buffer #2   2 min 
Staining buffer 20 min-30 min max.   

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
6. Incubate with Ab + Blocking Buffer Cocktail Solution

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
6.3. Carefully dry slide around tissue with a Kimwipe™.
Then pipette 190 μL Ab cocktail solution onto slide to cover tissue section.
Avoiding pipetting directly onto tissue.
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
6.4. Incubate slides, covered in a lightly humidified chamber, for 180 min Room temperature . 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7. Post Stain Wash and Multiple Fixations 
Tissue slides are briefly washed in staining buffer followed by sequential fixation with paraformaldehyde, ice-cold methanol, and final fixation solution.

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.1. Incubate in sequential Coplin Jars containing:
 Staining Buffer #1. 2 min
 Staining Buffer #2.  2 min 
 1.6% paraformaldehyde (Diluted in Storage buffer, Akoya Biosciences from 20% stock). 10 min 
Rinse slides sequentially in 3 Coplin Jars containing 1 x PBS (3 dips each) 
pre-chilled, -20 °C, methanol on ice  5 min  
Rinse slides sequentially in 3 Coplin Jars containing 1 x PBS (3 dips each) 

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.2. Prepare Final Fix 2% dilution
20 μl aliquot of Final Fix (Akoya Biosciences) diluted in 1 ml PBS

Carefully dry slide around tissue with a Kimwipe™ 
Then pipette 190 μL of 2% Final Fix solution  onto slide to cover tissue section while avoiding pipeting directly onto tissue. 
Incubate 20 min .

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.3.  Rinse slides sequentially in 3 Coplin Jars (3 dips each) PBS

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
8. Photobleaching and Storage

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
8.2. In order to reduce autofluorescence from the tissue, on the day prior to imaging, lay the slide to be imaged flat in a 100 cm2 dish containing Storage Buffer (Akoya Biosciences).
Photobleach by illumination with a 200 mA, 15 watts, 1600 lumens bulb 4 °C  .

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
8.1. Slides may be stored for up to 5 days in a Coplin Jar containing Storage buffer4 °C.

10. Prepare 1x Buffer Phenocycler Buffer and Reporter Stock Solution 
Reporter Stock Solution is prepared according to guidelines in the attached Phenocycler-Fusion User Guide (Akoya Biosciences®)
11. Prepare Reporter Solutions for each cycle in an opaque or foil wrapped tube.
Keep Reporters on ice. 
Centrifuge Reporter stock tubes briefly prior to removing aliquot.
Protect the fluorescent dye-containing Reporters from photobleaching.
Avoid introducing bubbles into the solutions.
Important to keep the reporter plate dust-free.

An imaging cycle = 4 channel detection including nuclear stain and 3 reporters, depending on filter set chosen, for example one each of ATTO550, Cy5/AF647, AF750. A multi-cycle experiment composes an imaging run.
SECTION: Lung section  preparation
1.1. FFPE Lung sections are prepared as described in dx.doi.org/10.17504/protocols.io.kxygxejwdv8j/v2 and mounted on Leica® Surgipath Apex Superior Adhesive Slides. 
None. 
    
SECTION: Multiplexed Imaging and Analysis
12. Image Acquistion via Phenocycler-Fusion® (i.e., CODEX V2)
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12.8. Images were acquired utilizing the 20X (0.5 µM/pixel) objective and Fusion 1.0.8 software.
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12.9. Image processing is automated via the Fusion 1.0.8 software and completed at the end of the experiment run.
 

SECTION: Multiplexed Imaging and Analysis
12.11. Rapid review of the resultant image.qptiffs is performed utilizing PhenoChart 1.2.0 software.  If necessary, exposure time (ms) is adjusted in the Phenocycler Experiment Designer to obtain readily detectable, specific marker signals that are below saturation.  Note that after viewing qptiffs in Phenochart an_annotations.xml.lock file will appear in the image folder.
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15. Image Analysis and Segmentation
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15.1. Analysis of processed image.qptiff files is performed utilizing QuPath (See Reference 3).
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15.2. Cell segmentation based on (DAPI) stained nuclei is performed utilizing the respective StarDist extension (See Reference 4) in QuPath.
SECTION: Custom antibody conjugation
18. Custom Antibody Conjugation is performed as described dx.doi.org/10.17504/protocols.io.3fugjnw.    
SECTION: Custom antibody conjugation
18.1. For antibodies containing sodium azide (0.05-0.1%) or trehelose (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.
SECTION: Custom antibody conjugation
18.2. Success of Antibody-Barcode chemical conjugation is determined by resolving unconjugated and conjugated Ab's on BioRAD‱s MiniProtean TGX Gel 4-15% Bis-Tris Protein Gels in accordance with Guidelines in the Phenocycler-Fusion User Guide (Akoya Biosciences®, Malborough, MA).
 


SECTION: Reporter Plate and Experiment Design
9. Reporter plate design and Phenocycler-Fusion run protocols are developed using the PhenoCycler Experiment Designer Software (Akoya Biosciences).  
SECTION: H&amp;E staining Post Phenocycler-Fusion
19.  It is useful to stain the tissue sections with hematoxylin & eosin following the multiplexed fluorescence imaging in order to match histology with antibody staining. We have stained lung sections with an aqueous H&E stain as described dx.doi.org/10.17504/protocols.io.kqdg397yeg25/v1 but this resulted in an overall rather grey stain and so we have opted instead to remove the flow cell by soaking in xylene prior to H&E staining of lung sections as described in Step 19.1. 


SECTION: Multiplexed Imaging and Analysis
12.3. After photobleaching in storage buffer, wash slides in PBS (250 ml; Coplin Jar)Room temperature 10 min 

SECTION: Multiplexed Imaging and Analysis
12.2. If necessary, warm reporter plate to Room temperature 

SECTION: Multiplexed Imaging and Analysis
12.4. After the wash, dry the bottom of the slide and around the edges of the tissue with a kimwipe. 
Attach a flow cell using the flow cell assembly device (Akoya Biosciences).  Center and press the flow cell onto the slide for  30 s

SECTION: Multiplexed Imaging and Analysis
12.5. Cure the flow cell adhesive by incubating the slide in 1X phenocycler buffer (Akoya biosciences)10 min Room temperature 

SECTION: Multiplexed Imaging and Analysis
12.7. Start an imaging run by turning on the Phenocycler fluidics system and the Phenoimager, followed by launching the Fusion software.  Select Start experiment and follow the prompts.
SECTION: Multiplexed Imaging and Analysis
12.6. Fill respective Reagent reservoirs on the Phenocycler side car with DMSO, 1X phenocycler buffer, and ddH20, and place a blank flow cell in the flow cell carrier attached to the fluidics tubing.
SECTION: Multiplexed Imaging and Analysis
12.12. After the run return the tissue slide with attached flow cell to storage buffer 4 °C.  If necessary, slides can be reimaged with a new set of reporters up to 5 days post staining without loss of signal.

SECTION: Multiplexed Imaging and Analysis
12.10. Checking the Sample mask, BF overview, and FL overview, by dragging and dropping files into ImageJ after the first cycle is recommended.  If major issues are observed, the run may be aborted to preserve reporters.
Do not attempt to open raw or intermediate cycle.qptiff during the imaging run.

SECTION: Conversion of .qptiff files to ome.tiff for upload to Omero utilizing bfconvert
16. Download Command Line Tools from https://www.openmicroscopy.org/bio-formats/downloads/

SECTION: Conversion of .qptiff files to ome.tiff for upload to Omero utilizing bfconvert
17. Execute the following in command prompt:
SECTION: Conversion of .qptiff files to ome.tiff for upload to Omero utilizing bfconvert
17.1. C:\>Set BF_MAX_MEM=10240M
SECTION: Conversion of .qptiff files to ome.tiff for upload to Omero utilizing bfconvert
17.2. C:\>bfconvert -no-sas -series 0 -compression LZW -pyramid-resolutions 5 -pyramid-scale 2 INPUT.qptiff OUTPUT_ome.tiff
SECTION: Conversion of .qptiff files to ome.tiff for upload to Omero utilizing bfconvert
17.3. Upload the resulting ome.tiff file to the CODEX folder in Omero and the respective tissue donor subfolder (e.g. D265) utilizing the Omero importer.
SECTION: H&amp;E staining Post Phenocycler-Fusion
19.1. After all MxF imaging is completed for the slides, remove the flow cell covered slides from storage buffer (Akoya Biosciences), wipe dry with Kimwipe and, utilizing a vacuum-suction apparatus similar to what is described in dx.doi.org/10.17504/protocols.io.kqdg397yeg25/v1, remove buffer from the flow cell.  
Incubate slides in a Coplin Jar containing Xylene for up to  240 min in order to weaken or dissolve the flow cell adhesive.
Carefully pry flow cell off the slide with razor blade or preferably a thin spatula. 
Proceed with H&E staining according to the standard protocol described here dx.doi.org/10.17504/protocols.io.36wgqjnq3vk5/v4 beginning at Step 5.
SECTION: Multiplexed Imaging and Analysis
15.5. Vascular smooth muscle and airway epithelial features are annotated and classified by thresholding the ACTA2 and PanCK signals utilizing the Pixel classifier in QuPath.
SECTION: Multiplexed Imaging and Analysis
15.4. Cell segmentation masks are exported from QuPath running the following ExpCell script within Script Editor:
 


SECTION: Multiplexed Imaging and Analysis
15.6. Annotation masks are exported from QuPath running the following ExpCell script within Script Editor:
 


SECTION: Multiplexed Imaging and Analysis
15.7. Object Data is exported as a GeoJSON file.
SECTION: Multiplexed Imaging and Analysis
15.3. Cell detection measurements (e.g. X,Y coordinates, fluorescent intensity data for all channels) are saved as a text file, opened in Excel as a tab delimited file, and resaved as a CSV (comma delimited) file.
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
6.1. Wash slides in Coplin Jars containing dd H2O
dd H20 #1 - 3 dips
dd H20 #2 - immersion 2 min 

10.1. Prepare 1 L of 1 x Phenocycler Buffer with Buffer additive, sufficient for 10 imaging runs.  This is used in the side-car Phenocycler Buffer bottle, in the reporter stock solution and in the mounting of the flow cell.  Do not filter. Store at room temperature for up to 2 weeks. Combine in clean glass container, mix well, avoid bubbles:
800 mL ddH2O
100 mL 10x Buffer for Phenocycler (Akoya Biosciences)
100 mL Buffer Additive for Phenocycler Fusion (Akoya Biosciences)

10.2. Prepare Reporter Stock Solution
Combine, mix gently by inversion:
1 x Phenocycler Buffer with Buffer additive 270 µL 
Assay Reagent 25 µL 
Nuclear Stain 5 µL 

Total volume should equal 300 μl x N = number of cycles plus two blank cycles (# reporter plate wells) to be done in the imaging run.
11.2. Pipette 245 μl of each reporter mix into a well of a light-opaque 96 well microtiter plate (Akoya Biosciences) according to the Experimental Design developed in the experimental design app. 

Also, pipette 245 ul reporter stock solution (blanks) into two wells of row H to create 2 blank wells. Seal the solution-containing wells with Akoya foil seals. The plate may be stored @ 40C  for up to 14 days in accordance with the attached PhenoCycler-Fusion User Guide (Akoya Biosciences).
11.1. Label an opaque microtainer for each well, each well = one imaging cycle of the imaging run. To each tube add 5 μl of each predetermined reporters complementing the antibody barcodes to be detected in that cycle, and add the Reporter Stock Solution to make total volume 250 μl per microtainer. 
SECTION: Multiplexed Imaging and Analysis
12.1. Use the Experiment Designer app, located on the PhenoCycler-Fusion Acquisition PC desktop,
to define the design of the Reporter plate and imaging parameters to be used in the experiment.  Follow details provided in the attached PhenoCycler-Fusion User Guide.

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
2. Deparaffination
Paraffin wax is removed from lung sections by incubation in xylene (3X)
Xylene #1 5 min 
Xylene #2 5 min 
Xylene #3 5 min 
SECTION: Multiplexed Imaging and Analysis
13. Record Imaging Run MetaData
SECTION: Multiplexed Imaging and Analysis
13.1. Record Slide ID, staining and imaging date in the Metadata Excel file located in the Fusion folder on the Phenocycler PC.
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13.4.  The image acquisition time = length of time between time stamp on the MarkerList or CombineInputs files (START) and the time stamp on the Akoya whole slide scan file .qptiff (FINISH).
SECTION: Multiplexed Imaging and Analysis
13.3. The "source storage time" = length of time (Days) between slide staining date and imaging date.
SECTION: Data backup and storage
14. Upload files from Phenocycler-Fusion runs to the URMC local Bluehive-Archive/archive/lungmap_lab codex folder.  
SECTION: Data backup and storage
14.1.  Maintain directory structure for each run by creating a new folder labeled with the slide ID containing a scan# subfolder, and a temp subfolder within the scan folder. 
SECTION: Data backup and storage
14.2. Include the following files in the upload:
Slide folder:
    Scan Folder:
     A. (.xpd) file (Phenocycler Experiment designer)
     B. Akoya whole slide scan .qptiff
     C. The ome.tiff generated via bfconvert (See step 17)
      Temp folder:
         i. CombineInputs
         ii. Coverslip Mask
         iii. qptiff raw files for each cycle
         iv. qptiff.intermediate files for each cycle
         v. FocusMap
         vi. FocusTable
         vii. Label
         viii. MarkerList
         ix. Overview BF
         x. Overview FL
         xi. SampleMask
SECTION: Multiplexed Imaging and Analysis
13.2. Likewise record the dates for biweekly Brightfield reference calibration and maintenance washes as well as monthy fluorescent and phenocycler reference calibration.
SECTION: Multiplexed Imaging and Analysis
13.5. The time_since_acquisition_instrument_calibration_value is the time interval (days) between the phenocycler reference calibration date and the imaging run date.