TITLE:  Multiplexed Immunofluorescence Staining and Imaging of Lung Sections
AUTHORS: Jeffrey Purkerson

[DESCRIPTION]
This protocol describes multiplexed immunofluorescent 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 Phenocycler-Fusion User Guide provided by Akoya (2), and includes sections that describe A) lung tissue preparation, B) Integration of multiomic analysis (e.g., MALDI-Mass Spectroscopy) of lung tissue section into the Multiplex immunofluorescent staining protocol, C) Labeling of lung tissue sections with antibody-barcode conjugates, D) Reporter plate and experiment design, E) Multiplexed imaging and analysis, F) 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.
SECTION: Lung section  preparation
1.2. Heat Slide in oven at 60 °C   .
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
3. Deparaffination and Rehydration
Incubate slides (5 min) in Coplin Jars containing Xylene (3X) followed by a descending ethanol series followed by molecular biology grade distilled water (2X).
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
3.1. Xylene #1- 5 min 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
3.2. Xylene #2 5 min 

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
3.3. Xylene #3 5 min 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
3.4. 100% Ethanol #1 5 min 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
3.5. 100% Ethanol #2 5 min 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
3.6. 90% Ethanol 5 min 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
3.7. 70% Ethanol 5 min 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
3.8. 50% Ethanol 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
3.9. 30% Ethanol 5 min 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
3.10. ddH20 5 min 

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
3.11. ddH205 min 

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 and cover entire Coplin 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 with 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 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.

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
5. Prepare Antibody 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 (Vol. in μL)



Component            2 Slides            5 slides 

Staining Buffer   362 µL 905 µL 
N Blocker           9.5 µL   23.75 µL         
G Blocker           9.5 µL   23.75 µL 
J Blocker            9.5 µL   23.75 µL 
S Blocker            9.5 µL   23.75 µL 
Total Volume      400 µL  1000 µL 


 

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
5.2.  Pipette volume of blocking buffer corresponding to total buffer volume minus volume of antibodies to be added to blocking buffer in a 1.5 ml microfuge tube (See Table below). The Blocking buffer volume must be 60% of total antibody buffer volume for effective blocking.  If needed reduce staining buffer volume (µL) to achieve 60% blocking buffer volume in Ab solution.

 
     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
6.1. Immerse Slides in sequential Coplin jars containing the following buffers from Akoya Biosciences:
Hydration buffer   2 min
Hydration buffer   2 min 
Staining buffer 20 min-30 min max.   


SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
6. Wash Slides and Incubate with Ab Solution
Remove slides from cooled AR9 buffer and rinse briefly by dipping slides(3X) in Coplin Jar ddH20 followed by immersion in a second Coplin Jar ddH20  2 min .

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
6.2. Carefully dry slide around tissue with a Kimwipe™ and then pipette 190 μL Ab solution onto slide to cover tissue section while avoiding pipetting directly onto tissue.

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
6.3. Incubate slides covered in a humidified chamber for 180 min Room temperature . 

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7. Post Stain Wash-Fixation 
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 Coplin Jar #1 containing Staining buffer 2 min 

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.2. Incubate in Coplin Jar #2 containing Staining Buffer  2 min 

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.3. Incubate slides in Coplin Jar containing 1.6% paraformaldehyde (Diluted from 20% stock) 10 min 

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.4. Rinse slides sequentially in 3 Coplin Jars (3 dips each) containing PBS. 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.5. Incubate slides in Coplin jar on ice containing pre-chilled (-20 °C methanol5 min  

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

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.7. Carefully dry slide around tissue with a Kimwipe® and then pipette 190 μL Final Fix solution (20 ul of aliquot of final fix (Akoya Biosciences) diluted in 1 ml PBS onto slide to cover tissue section while avoiding pipetting directly onto tissue. Incubate 20 min 

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.8. 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
8.1. Prior to imaging the next day immerse a slide in a 100 cm2 dish containing Storage Buffer (Akoya Biosciences), and photobleached by illumination with a 200 mA, 15 watts, 1600 lumens bulb 4 °C  .

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

10. Prepare Reporter Stock Solution 
Report Stock Solution is prepared according to guidelines in the  Phenocycler-Fusion User Guide (Akoya Biosciences®)
11. Prepare Reporter Solutions for each cycle
 Cycle (N=# of imaging runs)
 

SECTION: N-glycan analysis prior to labeling with antibody-barcode conjugates
2. Slide Preparation for MALDI Mass Sprectrometry includes the following steps
SECTION: N-glycan analysis prior to labeling with antibody-barcode conjugates
2.1. Deparaffinization in Xylene (See Steps 3.1-3.3)

SECTION: N-glycan analysis prior to labeling with antibody-barcode conjugates
2.2. Delipidation in Ethanol and Rehydration (See Step 3.4-3.11)
SECTION: N-glycan analysis prior to labeling with antibody-barcode conjugates
2.3. Antigen Retrieval via immersion of slides in a Coplin jar containing 50mM citraconic buffer pH 3 and heating in a vegetable steamer 100 °C 30 min
SECTION: N-glycan analysis prior to labeling with antibody-barcode conjugates
2.4. Washing in Water (See Step 6)
SECTION: N-glycan analysis prior to labeling with antibody-barcode conjugates
2.5. Spraying Enzyme for N-Glycan Removal

SECTION: N-glycan analysis prior to labeling with antibody-barcode conjugates
2.6. Spraying Maldi-Matrix in 50% Acetonitrile
SECTION: N-glycan analysis prior to labeling with antibody-barcode conjugates
2.10. Preparation of slides for staining post MALDI Analysis begin at step 3.4 below.
SECTION: N-glycan analysis prior to labeling with antibody-barcode conjugates
2.7. MALDI Analysis (PNNL-protocol)
SECTION: N-glycan analysis prior to labeling with antibody-barcode conjugates
2.8. Removal of MALDI matrix with 50% Acetonitrile 2X for 2 min 

SECTION: N-glycan analysis prior to labeling with antibody-barcode conjugates
2.9. Slides are air-dried and shipped for multiplexed immunofluorescence staining. 
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. 
SECTION: Reagent/Supply List
None. H&E staining Post Phenocycler-Fusion imaging was performed as described dx.doi.org/10.17504/protocols.io.kqdg397yeg25/v1
None. 
    
SECTION: Multiplexed Imaging and Analysis
12. Image Acquistion via Phenocycler-Fusion (i.e., CODEX V2)
SECTION: Multiplexed Imaging and Analysis
12.7. Images are acquired utilizing the 20X (0.5 µM/pixel) objective and Fusion 1.0.8 software.
SECTION: Multiplexed Imaging and Analysis
12.8. 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
12.10. Rapid review of the resultant image.qptiffs was performed utilizing PhenoChart 1.2.0 software.  If necessary, exposure time (ms) was adjusted in the Phenocycler Experiment Designer to obtain readily detectable, specific marker signals that are below saturation.
SECTION: Multiplexed Imaging and Analysis
13. Image Analysis and Segmentation
SECTION: Multiplexed Imaging and Analysis
13.1. Analysis of processed image.qptiff files is performed utilizing QuPath.
SECTION: Multiplexed Imaging and Analysis
13.2. Cell segmentation based on DAPI stained nuclei is performed utilizing the respective StarDist extension (i.e., 0.3 or 0.4) in QuPath.
SECTION: Custom antibody conjugation
14. Custom Antibody Conjugation is performed as described dx.doi.org/10.17504/protocols.io.3fugjnw.    
SECTION: Custom antibody conjugation
14.1. For antibodies containing   (0.05-0.1%) or   (5%) buffer exchange is 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
14.2. 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
15. Slides with lung tissue sections covered with a flow cell  (See Phenocycler-User Guide) are stained with H&E as described dx.doi.org/10.17504/protocols.io.kqdg397yeg25/v1
Eosin staining tends to be less intense, therefore recommended duration of eosin staining is  10 min or longer.

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

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

SECTION: Multiplexed Imaging and Analysis
12.3. After the wash dry the bottom of the slide and around the edges of the tissue with a kimwipe and attach a flow cell using the flow cell assembly device (Akoya Biosciences).30 s 

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

SECTION: Multiplexed Imaging and Analysis
12.6. 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
12.5. Fill respective Reagent reservoirs on the Phenocycler side car with DMSO, 1X phenocycler buffer, and ddH20, and place a blank flow cell in the attached flow cell carrier.
SECTION: Multiplexed Imaging and Analysis
12.11. After the run return the slide 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
12.9. 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.