TITLE: 811.1 CODEXV1 Staining and Imaging Protocol
AUTHORS: Jeffrey Purkerson, Gloria S Pryhuber, heidie_huyck

[DESCRIPTION]
This protocol describes multiplexed immunofluorescent staining and imaging of FFPE lung tissue sections utilizing the Phenocycler-Keyence 800 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 CODEX® User Manual Rev C provided by Akoya (2) and includes sections that describe A) coating of coverslips with Poly-L-Lysine, B) Lung section preparation C) Labeling of lung tissue sections with antibody-barcode conjugates, D) Multiplexed Imaging with the Keyence BZ-X810 microscope, E) 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.  Images produced utilizing this protocol are published in reference 3.

[BEFORE_START]
Ensure all reagents, supplies, equipment needed to complete the protocol are available in sufficient quantities to complete the procedure.

[GUIDELINES]
An overview of Multiplexed Imaging using barcode conjugated antibodies can be found in the attached CODEX® User Manual Rev C provided, with permission, from Akoya.  Note, multiplexed immunofluorescent imaging with Phenyocycler-Keyence 800 has been supplanted by the Phenocycler-Fusion platform. 

[STEPS]
SECTION: Lung section preparation
2. Bake Sections to promote tissue adherence to coverslips.

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
3. Deparaffination and Rehydration
Incubate coverslips placed in a coverslip staining rack in Coppin Jars containing xylene (3X) followed by a descending ethanol series and finally by molecular biology grade distilled water (2X)
SECTION: Lung section preparation
2.2. Heat tissue sections on coverslips in a dry oven  60 °C     .

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
3.9. 30% Ethanol 5 min 

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
3.10. ddH205 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.  Antigen RetrievalpH 6 
Heat-Induced Epitope Retrieval (HIER) reverses protein crosslinking in FFPE tissue
SECTION: Lung section preparation
2.1. FFPE Lung sections are prepared as described in dx.doi.org/10.17504/protocols.io.kxygxejwdv8j/v2 except that tissue sections are placed on poly-L-lysine coated glass coverslip from previous section. Standard section thickness is 4-5 micron.
SECTION: Coat glass coverslips with poly-L-Lysine
1. Glass coverslips (22mm x 22 mm, #1.5, Thermo Fisher, Waltham) are acid-washed and coated with Poly-L-Lysine. 
SECTION: Coat glass coverslips with poly-L-Lysine
1.1. Acid-wash glass coverslip in glass beaker or petri dish containing 1 N HCl and place in an oven set to 50-60 C   with occasional agitation.

SECTION: Coat glass coverslips with poly-L-Lysine
1.2. Wash coverslip 4X with ddH20

SECTION: Coat glass coverslips with poly-L-Lysine
1.3. Rinse coverslips in 100% ethanol and dry them between sheets of Whatman filter paper. Store coverslips in a clean container.
SECTION: Coat glass coverslips with poly-L-Lysine
1.4. Prepare 1% Poly-L-Lysine stock solution by diluting 100 mg of Poly-L-Lysine powder into 10 mL  ddH2O. Prepare a working solution by diluting Poly-L-Lysine to 0.1% in ddH20.

SECTION: Coat glass coverslips with poly-L-Lysine
1.5. Place coverslips in a cover glass staining rack and immerse in a beaker containing 0.1% Poly-L-Lysine (Fischer Scientific, Waltham, MA) and incubate.  , minimum 720 minRoom temperature

SECTION: Coat glass coverslips with poly-L-Lysine
1.6. Wash coverslips via immersion in a series of 6 X 50 ml beakers containing ddH20; 3-5 dips in each beaker.

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
4.1. Dilute Antigen Unmasking Solution, Citric Acid Based (H-3300, Vector Lab, Burlingame, CA), AR6 buffer to 1/100 in ddH20 in 50 ml beaker. Immerse coverslips in buffer in staining rack. Wrap the jar containing the buffer and coverslips in aluminum foil.
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
4.2. Place beaker on rack in the InstantPot® pressure cooker 
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 beaker, partially unwrap foil without uncovering beaker, and allow coverslips 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 AR6 buffer and/or incubating in Staining buffer see below) and keep on ice.  Staining buffer and reagents were obtained from Akoya Biosciences (CODEX Staining kit P/N 7000008, Akoya Bioscience).
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
5.1. Component            2 Tissue Sections           

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

5.2. Pipette volume of blocking buffer corresponding to total buffer volume minus volume of antibodies to be added to blocking buffer (See Table below). Add antibodies to the blocking buffer as per table, including calculation for number of tissue sections to be stained.
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.

 

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
6. Wash Coverslip and Incubate with Ab Solution.




SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
6.2. With flat nose-forceps carefully remove coverslips from cooled AR6 buffer and transfer to wells of the 6 well-plate in the following sequence: 
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
6.1. Aliquot 4 ml Hydration buffer (Akoya Biosciences) and 4 ml Staining buffer (Akoya biosciences) into a 6 well plate.  2 wells/coverslip Hydration buffer and 1 well/coverslip Staining buffer/
SECTION: Coat glass coverslips with poly-L-Lysine
1.7. Store lysine-coated coverslips in a cool dry place and use within 2 months.
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
6.3. Carefully dab edge of coverslip on a Kimwipe to remove excess buffer and then place in humidity chamber and pipette 190 μL Ab solution onto cover tissue section.

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
6.4. Incubate coverslips in a humidity chamber for 180 minRoom 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. Into a 6 well plate, aliquot 4 ml Staining buffer (2 wells/coverslip)  and  1.6% paraformaldehyde freshly diluted (0.8ml 20% paraformaldehyde + 9.2 mL in Storage Buffer) (1 well/coverslip). 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.2. With flat nose-forceps transfer coverslip to Staining buffer well #1 2 min 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.3. With flat nose-forceps transfer coverslip to Staining buffer well #1 2 min 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.4. With flat nose-forceps transfer coverslip to 1.6% paraformaldehyde 10 min 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.5. Rinse coverslip sequentially 3X in 50 ml conicals containing 1xPBS (3 dips each). 

SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.6. Incubate coverslip on ice in a well containing 5 ml pre-chilled (-20 °C methanol5 min  
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.7. Rinse coverslip sequentially 3X in 50 ml conicals containing 1xPBS (3 dips each). 
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.8. Fixative Reagent (Akoya) tubes are single use only, do not re-freeze, finger thaw, only one tube (20ul) placed in 1 ml PBS per 5 slides. 
With flat-nose forceps carefully dab coverslip on a Kimwipe to remove excess buffer and then place in humidity chamber. Pipette 190 μL diluted fixative reagent onto slide to cover tissue section 20 min.
SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
7.9. Rinse coverslip sequentially 3X in 50 ml conicals containing 1xPBS (3 dips each). 
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. On the evening prior to imaging immerse the coverslip in 2-3 ml Storage buffer (Akoya Biosciences) in a 6-well plate and photobleach by illumination with a 200 mA, 15 watts, 1600 lumens bulb 4 °C  .


SECTION: Labeling of lung tissue sections with antibody-barcode conjugates
8.2. Coverslips may be stored for up to 5 days in a 6-well plate containing Storage buffer.
SECTION: Multiplexed Imaging with the Keyence BZ-X810 microscope
9. Detailed instructions for setting up CODEX® experiment runs utilizing the Keyence BZ-X810 microscope can be found in the Keyence 800 QRC RevE documentation provided by Akoya Biosciences.
SECTION: Custom Conjugation
11. Custom Antibody Conjugation is performed as described dx.doi.org/10.17504/protocols.io.3fugjnw.    
SECTION: Custom Conjugation
11.1. For antibodies containing sodium azide (NaN3) (0.05-0.1%) or Trehalose (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 Conjugation
11.2. Success of Antibody-Barcode chemical conjugation is determined by resolving unconjugation 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: Multiplexed Imaging with the Keyence BZ-X810 microscope
9.2. Mount Coverslip a Keyence stage insert and stain with DAPI (14.3 µM) for 3-5 min to facilitate identification of a region of interest (ROI). 

SECTION: Multiplexed Imaging with the Keyence BZ-X810 microscope
9.1. Set up the Keyence BZ-X810 inverted epifluorescence microscope with : A)  Keyence Pan Fluorite 4X/0.13 NA and Nikon® 20x Plan Fluorite 0.75 NA objectives, B) four filter cubes including CH1: DAPI (Ex 350 Em 461), CH2: Cy3/R ATT0550 (Ex545 Em 605), CH3: Cy5 AF633(Ex 632 Em 647), CH4: Cy7 AF750 (Ex 743 Em 767), and interface with a Phenocycler® fluidics system (Akoya Biosciences).  

SECTION: Multiplexed Imaging with the Keyence BZ-X810 microscope
9.3. Once an ROI is identified with the 4X objective, image 10-30 Z planes
within the ROI with the 20X objective over 8 cycles with the indicated reporters
and exposure times for respective channels.   
  
     Cycle #      Reporter 1 (Cy3)      Reporter 2 (Cy5)      Reporter
  3 (Cy7)        1      Blank      Blank      Blank        2      E-Cad – BX014      CD4 – BX003      CD20 – BX007        3      COL1A1 – BX054      CD68 – BX015      CD31 – BX001        4      Ki67 – BX047      CD45 – BX021      CXCL4 – BX004        5      KRT14 – BX002      HLA-DR – BX033      VIM – BX046        6      ACTA2 – BX052      CD1c – BX042      LYVE1 – BX025        7      SFTPC – BX020      CD3e – BX045      PanCK – BX019        8      Blank      Blank      Blank        Exposure* (ms)      250      335      500   


 
SECTION: Multiplexed Imaging with the Keyence BZ-X810 microscope
10. Image Processing (i.e. background subtraction, deconvolution, extended depth of field, shading correction, full stitching and diagnostic output) of Z-plane tile stacks was performed with Codex Processor 1.8.3.14 software (Akoya Biosciences).