Bake Sections to promote tissue adherence to the slide.

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.

Heat Slide in oven at 60°C .

Slide Preparation for MALDI Mass Sprectrometry includes the following steps

Deparaffinization in Xylene (See Steps 3.1-3.3)

Preparation of slides for staining post MALDI Analysis begin at step 3.4 below.

Delipidation in Ethanol and Rehydration (See Step 3.4-3.11)

Antigen Retrieval via immersion of slides in a Coplin jar containing 50mM citraconic buffer pH 3 and heating in a vegetable steamer 100°C 0h 30m 0s

Washing in Water (See Step 6)

Spraying Enzyme for N-Glycan Removal

Spraying Maldi-Matrix in 50% Acetonitrile

MALDI Analysis (PNNL-protocol)

Removal of MALDI matrix with 50% Acetonitrile 2X for 0h 2m 0s

Slides are air-dried and shipped for multiplexed immunofluorescence staining.

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).

Xylene #1- 0h 5m 0s

ddH20 0h 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

High pH Antigen Retrieval 9

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

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)

Place Coplin Jar in an InstantPot® pressure cooker with ddH20 to 1/3-1/2 depth of Coplin Jar.

Heat on high pressure setting 0h 20m 0s

After releasing pressure remove Coplin Jar, partially unwrap foil without uncovering slides and allow slides 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 AR9 buffer and/or incubating in Staining buffer see below) and keep on ice.

Table 1. Blocking Buffer Component Table (Vol. in μL)

Component 2 Slides 5 slides

Staining Buffer 362µL 905µL

N Blocker 9.5µL 23.75µL

G Blocker 9.5µL 23.75µL

J Blocker 9.5µL 23.75µL

S Blocker 9.5µL 23.75µL

Total Volume 400µL 1000µL

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.

A	B	C	D	E
SMA	Akoya	4450049	BX013	1:200
PanCK	Akoya	4450020	BX019	1:200
MPO	Akoya	4250083	BX098	1:200
Ki67	Akoya	4250019	BX047	1:200
Keratin5	Akoya	4450090	BX101	1:200
HLADR	Akoya	4550118	BX033	1:200
FOXP3	Akoya	4550071	BX031	1:200
ColIV	Akoya	4550122	BX042	1:200
CD8	Akoya	4250012	BX026	1:200
CD68	Akoya	4550113	BX015	1:200
CD45	Akoya	4550121	BX021	1:200
CD4	Akoya	4550112	BX003	1:200
CD3e	Akoya	4550119	BX045	1:200
CD31	Akoya	4450017	BX001	1:200
CD20	Akoya	4450018	BX007	1:200
CD163	Akoya	4250079	BX069	1:200
CD14	Akoya	4450047	BX037	1:200
CD11c	Akoya	4550114	BX024	1:200
E-Cadherin	Akoya	4250021	BX014	1:200
TPSAB1*	Abcam	ab2378	BX041	1:1000
SFTPC*	Invitrogen	PA5-71842	BX020	1:500
SCGB1A1*	R&D System	MAB4218	BX043	1:400
β-III-Tubulin*	R&D Systems	MAB1195	BX055	1:400
ENDRB*	R&D Systems	MAB4496	BX027	1:50
SCEL*	Abcepta	Abcepta	BX052	1:100
RAGE*	Abcam	ab228861	BX028	1:100
LYVE1*	R&D Systems	AF2089	BX025	1:100
COL1A1*	Abcam	ab88147	BX054	1:100
CD298*	Abcam	ab167390	BX005	1:100
CD1c*	Novus	ab156708	BX016	1:50
SCGB3A2*	Abcam	ab240255	BX002	1:400
TP63*	Abcam	ab214790	BX006	1:100
MUC5AC*	Abcam	ab212636	BX040	1:100
PROX1*	R&D Systems	AF2727	BX050	1:200
CXCL4*	Peprotech	500-P05	BX004	1:200

Whenever possible barcodes and reporters were assigned to specific antibodies based on predicted antigen abundance and relative channel sensitivity in accordance with the PhenoCycler-Fusion User Guide (Akoya Biosciences). *Denotes custom-conjugated antibody. Refer to custom-conjugated section at the end of the protocol.

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 0h 2m 0s .

Immerse Slides in sequential Coplin jars containing the following buffers from Akoya Biosciences:

Hydration buffer 0h 2m 0s

Hydration buffer 0h 2m 0s

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

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.

Incubate slides covered in a humidified 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.

Incubate in Coplin Jar #1 containing Staining buffer 0h 2m 0s

Incubate in Coplin Jar #2 containing Staining Buffer 0h 2m 0s

Incubate slides in Coplin Jar containing 1.6% paraformaldehyde (Diluted from 20% stock) 0h 10m 0s

Rinse slides sequentially in 3 Coplin Jars (3 dips each) containing PBS.

Incubate slides in Coplin jar on ice containing pre-chilled (-20°C methanol0h 5m 0s

Rinse slides sequentially in 3 Coplin Jars (3 dips each) PBS.

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 0h 20m 0s

Rinse slides sequentially in 3 Coplin Jars (3 dips each) PBS

Photobleaching and Storage

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 .

Slides may be stored for up to 5 days in a Coplin Jar containing Storage buffer 4°C .

Reporter plate design and Phenocycler-Fusion run protocols are developed using the PhenoCycler Experiment Designer Software (Akoya Biosciences).

Prepare Reporter Stock Solution

Report Stock Solution is prepared according to guidelines in the Phenocycler-Fusion User Guide (Akoya Biosciences®)

Prepare Reporter Solutions for each cycle

Cycle (N=# of imaging runs)

A	B	C	D
235 X N	5 ul X N	5 ul X N	5 ul X N
235 X N	5 ul X N	5 ul X N	5 ul X N
235 X N	5 ul X N	5 ul X N	5 ul X N

245 uL of reporter stock solution (blanks) or 245 uL reporter mix are aliquoted into light opaque microtiter plates, sealed, and stored @ 4 C in for up to 14 days in accordance with the PhenoCycler-Fusion User Guide (Akoya Biosciences)

Image Acquistion via Phenocycler-Fusion (i.e., CODEX V2)

If necessary, warm reporter plate to Room temperature

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.

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.

After photobleaching in storage buffer wash slides in PBS (250 ml; Coplin Jar)Room temperature

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).0h 0m 30s

Cure the flow cell adhesive by incubating the slide in 1X phenocycler buffer (Akoya biosciences)0h 10m 0s Room temperature

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.

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.

Images are acquired utilizing the 20X (0.5 µM/pixel) objective and Fusion 1.0.8 software.

Image processing is automated via the Fusion 1.0.8 software and completed at the end of the experiment run.

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.

Image Analysis and Segmentation

Analysis of processed image.qptiff files is performed utilizing QuPath.

Cell segmentation based on DAPI stained nuclei is performed utilizing the respective StarDist extension (i.e., 0.3 or 0.4) in QuPath.

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 (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.

Success of Antibody-Barcode chemical conjugation is determined by resolving unconjugated 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).

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 0h 10m 0s or longer.