811.1 CODEXV1 Staining and Imaging Protocol

Glass coverslips (22mm x 22 mm, #1.5, Thermo Fisher, Waltham) are acid-washed and coated with Poly-L-Lysine.

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.

Wash coverslip 4X with ddH20

Rinse coverslips in 100% ethanol and dry them between sheets of Whatman filter paper. Store coverslips in a clean container.

Prepare 1% Poly-L-Lysine stock solution by diluting 100 mg of Poly-L-Lysine powder into 10mL ddH2O. Prepare a working solution by diluting Poly-L-Lysine to 0.1% in ddH20.

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 12h 0m 0s``Room temperature

Wash coverslips via immersion in a series of 6 X 50 ml beakers containing ddH20; 3-5 dips in each beaker.

Store lysine-coated coverslips in a cool dry place and use within 2 months.

Bake Sections to promote tissue adherence to coverslips.

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.

Heat tissue sections on coverslips in a dry oven 60°C .

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)

Xylene #1- 0h 5m 0s

ddH200h 5m 0s

ddH200h 5m 0s

Xylene #2 0h 5m 0s

Xylene #3 0h 5m 0s

100% Ethanol #1 0h 5m 0s

100% Ethanol #2 0h 5m 0s

90% Ethanol 0h 5m 0s

70% Ethanol 0h 5m 0s

50% Ethanol 0h 5m 0s

30% Ethanol 0h 5m 0s

Antigen Retrieval 6

Heat-Induced Epitope Retrieval (HIER) reverses protein crosslinking in FFPE tissue

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.

Place beaker on rack in the InstantPot® pressure cooker

Heat on high pressure setting 0h 20m 0s

After releasing pressure remove beaker, partially unwrap foil without uncovering beaker, and allow coverslips to cool for a minimum of 1h 0m 0s . Attempting to rinse/wash without allowing slides to cool may reduce tissue adherence.

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).

Component 2 Tissue Sections

Staining Buffer 362µL

N Blocker 9.5µL

G Blocker 9.5µL

J Blocker 9.5µL

S Blocker 9.5µL

Total Volume 400µL

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.

A	B	C	D	E	F
Ki67	Akoya	4250019	BX047	1:200	1
HLADR	Akoya	4550118	BX033	1:200	1
CD68	Akoya	4550113	BX015	1:200	1
CD45	Akoya	4550121	BX021	1:200	1
CD4	Akoya	4550112	BX003	1:200	1
CD3e	Akoya	4550119	BX045	1:200	1
CD31	Akoya	4450017	BX001	1:200	1
CD20	Akoya	4450018	BX007	1:200	1
PanCK	Akoya	4450020	BX019	1:200	1
KRT14	Akoya	4450031	BX002	1:200	1
ECAD	Akoya	4250021	BX014	1:200	1
Vimentin*	Biolegend	677802	BX046	1:200	1
SFTPC*	Invitrogen	PA5-71842	BX020	1:200	1
LYVE1*	R&D Systems	AF2089	BX025	1:40	5
Col1A1*	Abcam	ab88147	BX054	1:200	1
CD1c*	Novus	ab156708	BX016	1:200	1
ACTA2*	Abcam	ab240654	BX052	1:2000	0.1
CXCL4*	Peprotech	500-P05	BX004	1:50	4
				Total Ab Vol	24.1

Whenever possible barcodes and reporters are assigned to specific antibodies based on predicted antigen abundance and relative channel sensitivity in accordance with the CODEX® User Manual Rev C (Akoya Biosciences, Marlborough, MA). * Denotes custom-conjugated antibody. Refer to custom-conjugation method section at the end of the protocol.

Wash Coverslip and Incubate with Ab Solution.

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/

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 0h 2m 0s

Hydration buffer 0h 2m 0s

Staining buffer 0h 20m 0s-0h 30m 0s max .

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.

Incubate coverslips in a humidity chamber for 3h 0m 0s``Room temperature

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.

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).

With flat nose-forceps transfer coverslip to Staining buffer well #1 0h 2m 0s

With flat nose-forceps transfer coverslip to Staining buffer well #1 0h 2m 0s

With flat nose-forceps transfer coverslip to 1.6% paraformaldehyde 0h 10m 0s

Rinse coverslip sequentially 3X in 50 ml conicals containing 1xPBS (3 dips each).

Incubate coverslip on ice in a well containing 5 ml pre-chilled (-20°C methanol0h 5m 0s

Rinse coverslip sequentially 3X in 50 ml conicals containing 1xPBS (3 dips each).

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 0h 20m 0s.

Rinse coverslip sequentially 3X in 50 ml conicals containing 1xPBS (3 dips each).

Photobleaching and Storage

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 .

Coverslips may be stored for up to 5 days in a 6-well plate containing Storage buffer.

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.

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).  

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).

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.   

A	B	C	D
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

*DAPI channel exposure ranged from 4-8 ms.

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).

Custom Antibody Conjugation is performed as described dx.doi.org/10.17504/protocols.io.3fugjnw. dx.doi.org/10.17504/protocols.io.3fugjnw.

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.

Success of Antibody-Barcode chemical conjugation is determined by resolving unconjugation and conjugated Ab's on BioRAD^TMs MiniProtean TGX Gel 4-15% Bis-Tris Protein Gels in accordance with Guidelines in the Phenocycler-Fusion User Guide (Akoya Biosciences®, Malborough, MA).