Xenium Prime In Situ Gene Expression (5K human or mouse panel+100 custom panel)

Xiangwei Fang; Feng Chen; Li Ding

Jun 26, 2026

Version 2

Xenium Prime In Situ Gene Expression (5K human or mouse panel+100 custom panel) V.2

DOI

https://dx.doi.org/10.17504/protocols.io.8epv5w144v1b/v2

Xiangwei Fang¹,
Feng Chen¹,
Li Ding¹

¹Washington University in St. Louis

Washington University in St. Louis

Xiangwei Fang

Washington University in St. Louis, School of Medicine

DOI: https://dx.doi.org/10.17504/protocols.io.8epv5w144v1b/v2

Protocol Citation: Xiangwei Fang, Feng Chen, Li Ding 2026. Xenium Prime In Situ Gene Expression (5K human or mouse panel+100 custom panel). protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5w144v1b/v2Version created by Xiangwei Fang

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: June 26, 2026

Last Modified: June 27, 2026

Protocol Integer ID: 319860

Keywords: xenium prime in situ gene expression, resolution spatial transcriptomics platf..., multiplexed detection of rna transcript, spatial transcriptomics platform, performing xenium prime analysis, situ gene expression, xenium prime, xenium prime analysis, gene custom panel, subcellular resolution, rna transcript, cancer biology, detailed characterization of tissue arch..., tissue architecture, cell segmentation, including cancer biology, cellular heterogeneity, thousands of transcript, xenium prime in situ gene expression, resolution spatial transcriptomics platform, multiplexed detection of rna transcript, spatial transcriptomics platform, performing xenium prime analysis, situ gene expression, xenium prime, xenium prime analysis, gene custom panel, subcellular resolution, rna transcript, cancer biology, detailed characterization of tissue architecture, tissue architecture, cell segmentation, including cancer biology, cellular heterogeneity, gene, thousands of transcript

Abstract

Xenium Prime In Situ Gene Expression is a high-resolution spatial transcriptomics platform that enables multiplexed detection of RNA transcripts directly within intact tissue sections. This protocol describes the workflow for performing Xenium Prime analysis using either the 5K human or mouse panel combined with a 100-gene custom panel. The method includes tissue section preparation, histological quality assessment, assay setup,  in situ hybridization, cell segmentation,  instrument loading, imaging & decoding, and post Xenium H&E. By preserving spatial context while profiling thousands of transcripts at single-cell or subcellular resolution, Xenium Prime supports detailed characterization of tissue architecture, cellular heterogeneity, and cell–cell interactions. This protocol is applicable to a broad range of research applications, including cancer biology, developmental biology, and disease microenvironment studies, and provides a flexible framework for integrating predefined panels with user-defined targets of interest.

Protocol materials

Sodium hydrosulfiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #157953 
HematoxylinMerck MilliporeSigma (Sigma-Aldrich)Catalog #MHS16 
Bluing BufferDakoCatalog #CS70230-2 
SelecTech Eosin 515 PhloxineLeica BiosystemsCatalog #3801606 
Tween&trade; 20 Surfact-Amps&trade; Detergent SolutionThermo FisherCatalog #28320 
 Invitrogen™ Nuclease-Free Water (not DEPC-Treated) Invitrogen - Thermo FisherCatalog #AM9932 
Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8418 
KCl (2 M), RNase-freeThermo Fisher ScientificCatalog #AM9640G 

Tissue and Xenium slides Preparation

Tissue Screening.

Review clinical information for all samples. Store FFPE tissue blocks at 4°C.

Identify FFPE blocks of interest and perform hematoxylin and eosin (H&E) staining for tissue evaluation.

Assess RNA quality of selected FFPE blocks using the DV200 metric.

Select FFPE blocks that meet quality criteria for downstream spatial profiling analyses.

FFPE Tissue Sectioning & Section Placement.

Optimal water bath temperature and section floating time are critical for tissue section expansion. 39ºC is the recommended water bath temperature for most tissues in our experience. However, water bath temperature may need optimization based on the tissue type. Optimization should occur before utilization of a Xenium slide.

FFPE tissue blocks can be trimmed or scored according to ROI selection to fit multiple sections onto the
Sample Area.

Incubate FFPE blocks on the ice bath for 10-30 min. Set the microtome to 5 μm for sectioning. Place the sections on Xenium slide within the fiducials.

Dry tissue sections upright in a drying rack at room temperature until tissue is opaque and no water remains on top of or under the section.

Xenium slides drying and storage.

Dry Xenium slides overnight at room temperature.

Place the slides in a slide drying rack and incubate for 3 h at 42°C in an oven or thermal cycler.

Store the slide containing dry tissue sections at room temperature in a desiccator for up to 4 weeks.

Xenium Prime Reactions

Deparaffinization.

Place slides in a Section Dryer Oven and incubate uncovered at 60°C for 30 mins. Remove from the oven or thermal cycler and allow the slide to cool down to room temperature for 7 min.

Gently immerse slide in the Xylene Jar 1. Incubate for 10 min. Gently immerse slide in the Xylene Jar 2 and incubate for 10 min.

Gently immerse slide in the 100% Ethanol Jar 1 for 3 min. Gently immerse slide in the 100% Ethanol Jar 2 for 3 min.

Gently immerse slide in the 96% Ethanol Jar 1 for 3 min. Gently immerse slide in the 96% Ethanol Jar 2 for 3 min.

Gently immerse slide in the 70% Ethanol Jar for 3 min. Gently immerse slide in the Nuclease-free water Jar for 20 sec.

Remove any remaining nuclease-free water from the slide using a lint-free laboratory wipe. Dry back of slide and front of slide outside of Sample Area completely without touching or damaging the tissue sections. Place the slide in the cassette. Add 500 μl 1X PBS.

Decrosslinking.

Prepare Diluted Perm Enzyme B. Prepare Decrosslinking Buffer using Xenium FFPE Tissue Enhancer, Diluted Perm Enzyme B, and Urea.

Add 500 μl Decrosslinking Buffer along the side of the well to uniformly cover the tissue sections, without introducing bubbles. Tap Xenium Cassette gently to ensure uniform coverage.

Incubate at 80°C in the pre-heated thermal cycler for 30 mins, followed by 22°C for 10 mins.

Three PBS-T washes: Add 500 μl PBS-T. Incubate for 1 min at room temperature. Remove all PBS-T. 
Add 500 μl PBS-T.

Priming Hybridization.

Obtain priming oligos that have been thawed or equilibrated to room temperature. Preheat pre-designed and/or add-on custom oligos by incubating at 95°C for 2 min in a heat block or thermal cycler, followed by 1 min on ice. Maintain on ice.

Prepare Priming Hybridization Mix. Retrieve the assembled Xenium Cassette, and Remove all PBS-T. Gently place a Xenium Cassette Insert onto the Xenium Cassette v2 using forceps. Add 150 μl Priming Hybridization Mix along to the Xenium Cassette Insert using the cut-out to pipette solution under the insert to uniformly cover the tissue sections, without introducing bubbles.

Incubate at 50°C in the pre-heated thermal cycler for 1 hour and 30 mins.

Post Priming Hybridization Wash.

Retrieve Post-Priming Wash Buffer and incubate at 37°C for 5 min or until thawed completely. Vortex and centrifuge briefly. Maintain at room temperature. 

Remove the Xenium Cassette Lid v2. Carefully pipette 200 μl PBS-T into Xenium Cassette Insert Cut-out to float the insert. Carefully remove Xenium Cassette Insert with forceps. Discard the insert. Wash with 500 μl PBS-T for 1 min at room temperature, 2 times.

Add 500 μl Post-Priming Wash Buffer to the well. Incubate at 50°C in the pre-heated thermal cycler for 30 mins.

RNase Treatment.

Prepare RNase Mix on ice.

Three PBS-T washes: Add 500 μl PBS-T. Incubate for 1 min at room temperature. Remove all PBS-T.

Add 500 μl RNase Mix to the well. Incubate at 37°C in the pre-heated thermal cycler for 20 mins.

Polishing.

Prepare Polishing Reaction Mix on ice.

Three 0.5X SSC-T washes: Add 500 μl 0.5X SSC-T. Incubate for 1 min at room temperature.
Remove all SSC-T.

Add 500 μl Polishing Reaction Mix to the well. Incubate at 37°C in the pre-heated thermal cycler for 1 hour.

Probe Hybridization.

Obtain probes that have been thawed or equilibrated to room temperature. Immediately before use, preheat pre-designed and/or add-on custom probes by incubating at 95°C for 2 min in a heat block or thermal cycler, followed by 1 min on ice. Maintain on ice.

Three PBS-T washes: Add 500 μl PBS-T. Incubate for 1 min at room temperature. Remove all PBS-T.

Prepare Probe Hybridization Mix. Retrieve the Xenium Cassettes, and Remove all PBS-T. Gently place a Xenium Cassette Insert onto the Xenium Cassette v2 using forceps. Add 150 μl Probe Hybridization Mix along to the Xenium Cassette Insert using the cut-out to pipette solution under the insert to uniformly cover the tissue sections, without introducing bubbles.

Incubate at 50°C in the pre-heated thermal cycler Overnight (16 - 24 h).

Post Hybridization Wash.

Remove the Xenium Cassette Lid v2. Carefully pipette 200 μl PBS-T into Xenium Cassette Insert Cut-out to float the insert. Carefully remove Xenium Cassette Insert with forceps. Discard the insert. Wash with 500 μl PBS-T for 1 min at room temperature, 2 times.

Add 500 μl Post Hybridization Wash Buffer to the well. Incubate at 35°C in the pre-heated thermal cycler for 15 mins.

Ligation.

Prepare Ligation Mix shortly before using. Maintain on ice.

Three PBS-T washes: Add 500 μl PBS-T. Incubate for 1 min at room temperature. Remove all PBS-T.

Add 500 μl Ligation Mix to the well. Incubate at 42°C in the pre-heated thermal cycler for 30 mins.

Amplification.

Prepare Amplification Enhancer Master Mix on ice. Pipette mix 10X and centrifuge briefly. Maintain on ice.

Three PBS-T washes: Add 500 μl PBS-T. Incubate for 1 min at room temperature. Remove all PBS-T.

Immediately add 500 μl Amplification Enhancement Master Mix to the well. Incubate at 4°C in the pre-chilled thermal cycler for 2 hours.

Prepare Amplification Master Mix on ice. Maintain on ice.

Post Amplification Enhancement Wash: Remove the Xenium Cassette Lid v2. Using a pipette, remove all the buffer from the well. Add 500 μl Amplification Enhancer Wash Buffer to the well. Incubate 1 min at room temperature.

Remove all the buffer from the well. Immediately add 500 μl Amplification Master Mix (prepared in step 6.1) to the well. Incubate at 30°C in the pre-heated thermal cycler for 1 hour and 30 mins.

After Amplification is complete, immediately proceed to the next step. Three TE Buffer, pH 8.0 Washes: Add 500 μl TE Buffer, pH 8.0 to the well. Incubate 1 min at room temperature. Remove all TE buffer.

Cell Segmentation Staining (Multi-modal Segmentation is optional but Recommended)

Blocking.

Four ethanol washes: Add 1,000 μl 70% ethanol. Incubate for 2 min at room
temperature. Remove the ethanol. Add 1,000 μl 100% ethanol. Incubate for 2 min at room
temperature. Remove the ethanol. Add 1,000 μl 100% ethanol. Incubate for 2 min at room
temperature. Remove the ethanol. Add 1,000 μl 70% ethanol. Incubate for 2 min at room
temperature. Remove the ethanol.

Immediately add 1,000 μl PBS-T. Incubate for 1 min at room temperature. Remove all PBS-T. Add 500 μl 1X Diluted Xenium Block and Stain Buffer to cassette for blocking. Incubate for 1 hour at room temperature.

Primary staining.

During incubation, prepare Xenium Multi-Tissue Stain Mix. Incubate resuspended Xenium Multi-Tissue Stain Mix for 30 min at room temperature. Centrifuge Xenium Multi-Tissue Stain Mix for 10 min at 14,000 rcf at 4°C. Maintain on ice.

Remove the Xenium Cassette Lid v2 and using a pipette, remove 1X Diluted Xenium Block and Stain Buffer from well corners.Gently place Xenium Cassette Insert onto the Xenium Cassette using forceps.

Using a pipette along the side of the tube (avoid touching pellet), withdraw 100 μl Xenium Multi-Tissue Stain Mix and add to the Xenium Cassette Insert using the cut-out to pipette solution under the Xenium
Cassette Insert. Incubate the Xenium Cassette overnight (16-24 h) at 4°C (in a refrigerator, incubator, or in cold room).

Stain Enhancement.

Prepare Xenium Stain Enhancer.

Remove the Xenium Cassette Lid v2. Carefully pipette 200 μl PBS-T into Xenium Cassette Insert Cut-out to float the insert. Carefully remove Xenium Cassette Insert with forceps. Discard the insert. Wash with 500 μl PBS-T for 1 min at room temperature, 3 times.

Add 500 μl resuspended Xenium Stain Enhancer to the well. Incubate at room temperature for 20 min.

Three PBS-T washes: Add 500 μl PBS-T. Incubate for 1 min at room temperature. Remove all PBS-T. After Cell Segmentation Staining is complete, proceed immediately to the next step.

Autofluorescence Quenching and Nuclei Staining.

1h 15m

Autofluorescence Quenching.

Prepare diluted Reducing Agent B. Maintain at room temperature.

Add 500 μl Diluted Reducing Agent B, incubate for 10 min at room temperature.

Three ethanol washes: Add 1,000 μl 70% ethanol. Incubate for 1 min at room
temperature. Remove the ethanol. Add 1,000 μl 100% ethanol. Incubate for 1 min at room
temperature. Remove the ethanol. Add 1,000 μl 100% ethanol. Incubate for 1 min at room
temperature. Remove the ethanol.

Prepare Autofluorescence Solution. Maintain at room temperature.

Add 500 μl Autofluorescence Solution to the well. Incubate for 10 min at room temperature in the dark.

Three ethanol washes: Add 1,000 μl 100% ethanol. Incubate for 2 min at room
temperature. Remove the ethanol.

Place Xenium Cassette v2 without lid on the Thermocycler Adaptor v2 on
the thermal cycler to dry at 37°C for 5 mins. DO NOT close the thermal cycler lid.

Immediately remove the cassette from the Thermocycler Adaptor and place on a flat, clean work surface. Add 1,000 μl 1X PBS prepared at step 1.1 to rehydrate the tissue and incubate for 1 min at room temperature in the dark.

Nuclei Staining.

15m

Remove all 1X PBS. Add 1,000 μl PBS-T and incubate for 2 min at room temperature in the dark.

Remove all PBS-T from the well. Add 500 μl Nuclei Staining Buffer and incubate 1 min at room temperature in the dark.

Remove all Nuclei Staining Buffer. Three PBS-T washes: Add 1,000 μl PBS-T. Incubate for 1 min at room temperature in the dark. Remove all PBS-T.

Add 1,000 μl PBS-T. Store slides (Short-term storage for ≤1 week at 4°C in the dark) or alternatively, proceed directly to the Xenium Analyzer for scanning and decoding.

Xenium Analyzer for Scanning and Decoding

6d 4h 5m

Reagent Plate Preparation.

Reagent plates are packaged in mylar bags for protection. Keep plates in mylar bag during storage and thawing. When ready to use, open the bag and remove the foil-sealed plate prior to preparation for loading. Thaw Xenium Prime Decoding Reagent Module B - 5K at 4ºC for 16–72 h prior to handling and loading onto the instrument.

Take Xenium Decoding Reagent Module A out of the mylar packaging. Mix by gently inverting the plate 20X without introducing bubbles. DO NOT vortex. Place the reagent plate and counterbalancing plate in a swinging bucket centrifuge. Once balanced, centrifuge at 1600 rcf for 10 min at room temperature. Maintain on ice or 4ºC.

15m

Take Xenium Decoding Reagent Module B out of the mylar packaging. Equilibrate thawed plate at room temperature for 30 min. Mix by gently inverting the plate 20X without introducing bubbles. DO NOT vortex. Place the reagent plate and counterbalancing plate in a swinging bucket centrifuge. Once balanced, centrifuge at 300 rcf for 1 min at room temperature. Maintain on room temperature.

35m

Buffer Preparation.

Fill Reagent Bottle #1 with 500 ml Nuclease-free Water/Nuclease-free Milli-Q Water and cap bottle with standard bottle cap.

Prepare 1L 1X PBS-T each for Bottle #2 and Bottle #3. Maintain at room temperature. Cap bottle and invert gently to mix. Ensure minimal bubbles are introduced.

Prepare Probe Removal Buffer for Bottle #4. Add reagents in the order listed. Nuclease-free Water Invitrogen™ Nuclease-Free Water (not DEPC-Treated) Invitrogen - Thermo FisherCatalog #AM9932   232.5 ml; DMSODimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8418   250 ml; KClKCl (2 M), RNase-freeThermo Fisher ScientificCatalog #AM9640G   12.5 ml; 10% Tween-20Tween&trade; 20 Surfact-Amps&trade; Detergent SolutionThermo FisherCatalog #28320   5 ml. Cap bottle and invert gently to mix. Ensure minimal bubbles are introduced. Maintain at room temperature for 30 min to cool it down and to clear bubbles created during mixing. Minor amounts of bubbles are acceptable.

System Operation.

30m

Initialize Instrument. Click “Start New Run” button. System checks take ~ 10 min. Input Run Name. Select which 10x assay was used to prepare samples. Select Onboard Analysis Version. Add Cassette Details. Select Gene Panel.

Load Consumables, including: Reagent bottles with buffer (Reagent Preparation section), Xenium
Buffer Caps; Waste Bottle; Reagent Plates; Pipette Tip Rack; Extraction Tip; Objective Wetting Consumable; Waste Tip Tray; Cassette/s (with tissue sections on the Xenium Slide ready for the
instrument run).

Sample Scan: Instrument will begin Sample Scan. The scanned image will be used for region selection.

Region Selection: after initial overall scanning, click “Add Region” to designate imaging area(s) for each slide cassette.

45m

Initiate Run: depending on tissue type, region size, sample quality, etc, times ranges from 5 to 7 days.

Post-Xenium H&E Staining

3h 45m

Store analyzed Xenium Cassettes in PBS-T at 4°C following a Xenium Analyzer instrument run. Proceed to H&E Staining within 3 days following an instrument run.

Xenium Cassette Removal and Quencher Removal.

Remove slide from Xenium Cassette.

Immerse in Quencher Removal Solution (17.4 mg Sodium Hydrosulfite in 10 ml Milli-Q Water Sodium hydrosulfiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #157953   & incubate for 10 mins.

10m

Immerse in Milli-Q water & incubate for 1 min, 3 times.

Immerse in 1X PBS and store up to 2 days at 4°C.

H&E Staining.

Gently immerse slide in the Milli-Q Water Jar for 2 min at room temperature.

Gently immerse slide in the HematoxylinHematoxylinMerck MilliporeSigma (Sigma-Aldrich)Catalog #MHS16   Solution Jar for 20 min at room temperature.

20m

Gently immerse slide in the Milli-Q Water Jar for 1 min at room temperature, 3 times.

Gently immerse slide in the BluingBluing BufferDakoCatalog #CS70230-2   Solution Jar for 1 min at room temperature.

Gently immerse slide in the Milli-Q Water Jar for 1 min at room temperature.

Gently immerse slide in the 70% Ethanol Jar for 3 min at room temperature.

Gently immerse slide in the 95% Ethanol Jar for 3 min at room
temperature.

Gently immerse slide in the EosinSelecTech Eosin 515 PhloxineLeica BiosystemsCatalog #3801606   Solution Jar for 2 min at room
temperature.

Gently immerse slide in the 95% Ethanol Jar for 30 sec at room temperature, 2 times.

Gently immerse slide in the 100% Ethanol Jar for 30 sec at room temperature, 2 times.

Gently immerse slide in the Xylene Jar for 3 min at room temperature, 2 times.

Coverslipping.

Before mounting the coverslip, ensure that the slide is dry. Moisture on the surface of the slide may result in faulty mounting. Wipe away any residual droplets with a lint-free laboratory wipe.

Place slide on a flat, clean, non-absorbent work surface. Using a wide-bore pipette tip, add 150-200 μl mounting media to uniformly cover all tissue sections on the slide. Apply the coverslip at an angle on one end of the slide. Slowly lower the coverslip, without introducing bubbles. Allow mounting media to spread and settle.

Dry the coverslipped slide for 30 min at room temperature.

30m

Once coverslipping is complete, proceed with imaging. Samples may be stored temporarily at 4°C and imaged within 3 days after coverslipping. For long-term storage after imaging, maintain at room temperature in the dark.