Dec 13, 2025
Xenium Spatial Transcriptomics with Protein add-on Protocol for Human Tissue
- Sanjay Jain1,
- Amanda Knoten1,
- Tao Cheng1,
- kaushalm 1,
- Asmita Kishor Lagwankar1,
- Stephanie Reinert1
- 1Washington University, Saint Louis
- KPMP
- Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Protocol Citation: Sanjay Jain, Amanda Knoten, Tao Cheng, kaushalm , Asmita Kishor Lagwankar, Stephanie Reinert 2025. Xenium Spatial Transcriptomics with Protein add-on Protocol for Human Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx4q6zl8j/v1
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: October 21, 2025
Last Modified: December 15, 2025
Protocol Integer ID: 230332
Keywords: Deparaffinization, Hybridization, Amplification, Nuclei staining, Cell segmentation, Xenium, Spatial Transcriptomics, Kidney, xenium spatial transcriptomics protocol for human kidney, xenium spatial transcriptomics protocol, post xenium assay, performing spatial transcriptomic, spatial transcriptomic, using 10x xenium, human kidney, light microscopy evaluation, disease kidney, 10x xenium, light microscopy, flanking tissue section, situ imaging, tissue section, selecting tissue, assay performance, posy imaging, imaging, xenium spatial transcriptomics with protein, xenium spatial transcriptomic, protein marker panel, adding protein marker panel, 10x xenium with add, on of protein marker, protein marker, protocol for human tissue, marked cell
Funders Acknowledgements:
Common Fund_HuBMAP NIH/NIDDK
Grant ID: U54DK134301
KPMP NIH/NIDDK
Grant ID: 2U01DK114933
Disclaimer
This protocol is still in development and the analysis steps may change
Abstract
This is a protocol for performing spatial transcriptomics using 10X Xenium with add-on of protein markers. Here we are using custom panel designed to target cell types in the healthy and disease kidneys and discover their spatial relationships. The protocol has many steps involving selecting tissue, tissue sectioning and mounting on slides, processing to prepare for hybridization with custom panel and in situ imaging. This protocol also includes adding protein marker panels commercially available from 10X to further aid in identifying the marked cells and determine RNA and Protein overlap. Post imaging there are number of analyses that can be performed and these will depend on the biological questions where the methods and tools are still evolving, however, we will focus on key QC parameters to evaluate assay performance. Each of the steps from sectioning to data generation have quality assurance and control steps. Post Xenium assay, slides are stained with hematoxylin and eosin for light microscopy and flanking tissue sections are typically used for light microscopy evaluation or other technologies.
Image Attribution
The slide template is from 10X Genomics
Guidelines
Panel design: Prior to starting the assay make sure you have all the reagents for Xenium, here V1 kit is used. Also we are using custom panels that target genes of interest and these should be in hand already. These are designed based on specific markers from human kidney atlas, literature and validating them against various spatial transcriptomic technologies. Once this list is finalized, follow 10X guidelines to validate compatibility of probe designs and target genes using 10X tools and adjust for overcrowding based on 10X recommendations (https://www.10xgenomics.com/support/software/xenium-panel-designer/latest). The number of target genes can be modified up to the maximum 10X permits.
Materials
Reagents:
- Epredia™ Modified Harris HematoxylinFisher ScientificCatalog #22-050-206
- Eosin Y Solution, Alcoholic, with PhloxineMerck MilliporeSigma (Sigma-Aldrich)Catalog #HT110380
- Ammonium hydroxide solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #221228-500ML-A
- Hydrochloric acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #H1758-500ML
- Xylene, Fisherbrand™, HistoPrep™Fisher ScientificCatalog #HC-700-1GAL
- Acetic Acid, Glacial Fisher ScientificCatalog #A38S-500
- Ethanol (200 Proof) receiving dock Cat. # 11100020S
Kits
needed
Sample Prep Reagents: PN-1000460
Decoding Consumables: PN-1000487
Gene Panel:
Custom
10X
Protein panels
Xenium
Protein Immune Cell Subpanel A PN:
2001380
Xenium
Protein Immune Cell Subpanel B PN:
2001381
Xenium
Protein Immune Cell Subpanel C PN:
2001426
Xenium
Protein Immune Checkpoint Subpanel PN:2001383
Xenium
Protein Proliferation & Differentiation Subpanel PN:
2001382
Xenium
Protein Tumor Subpanel PN:
2001384
Troubleshooting
Safety warnings
If the instrument stops during the run and you get an error, please contact 10X support immediately as you may be able to salvage your run. If for some reason the instrument is to be stopped then carefully follow the protocol to remove the slides, keep them in buffer, and follow 10X advice on starting the run.
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Before start
For added protein, make sure you have all the compatible reagents and kits.
Tissue Preparation - Tissue selection
Note
We adopted the protocol suggested by 10X for tissue mounting on Xenium slides Detailed protocol here: Xenium FFPE Tissue Prep guide. If you are using an existing RNA only kit for dual RNA+Protein assay, make sure to get appropriate protein-compatible reagents from 10X to be able to perform both modalities
Perform the assay on FFPE sections. To ensure proper preservation of tissue ensure warm ischemia time is <4h and tissue is preserved in fixative as soon as possible (preferably within less than an hour from retrieval. For KPMP consortia, this is not a concern as most biopsies are preserved in less than 20 min.
Although the assay can work on a few mm of tissue, ideally the tissue should be 5mm or larger. If larger than 2cm in length will need to either trim it or bend it to ensure it will fit in the designated area on Xenium slide (QC).
Tissue Preparation - Sectioning
11h 30m
Equilibrate blank xenium slides to Room temperature for 00:30:00 .
30m
Trim the extra wax to be able to fit as many sections in the designated area on the Xenium slide (see picture 1) in a timely manner so the sections do not disintegrate on the water bath.
Cut a few sections from the surface to minimize surface contamination, use one of them for light microscopy to ensure presence of kidney tissue (QC). Once confirmed, cut and mount sections at 5uM on the xenium slide. If tissue of interest is already confirmed then can skip.
Center sections in the outlined scan area (10.45mm x 22.45mm)
- Fiducials just inside white outline
Picture 1
Do Not overlap tissues or paraffin.
- Do not place tissues over fiducials
- Try not to resubmerge previously mounted tissue when placing additional sections, see 10X tips in the link above.
Place tissues on etched label side of slide.
Bake slides at 42 °C for 03:00:00 , then at Room temperature Overnight .
11h
Store slides at Room temperature (with dessicant) until use.
- use within 4 weeks for best results.
Note
QC: Ensure mounted sections are not folded over, or fragmented or have extensive shatter
Assay
16h 37m 40s
Here we follow the protocol recommended by Xenium chrome-extension:
Deparaffinization, hydration and decrosslinking
Follow xenium protocol. For quality assurance, ensure that sections are completely immersed during these steps.
Note
QC: Check that the tissue has not lifted off the slides, if it has discard and start again
Custom panel addition, morphological marker staining, add-on protein and hybridization
Probe hybridization. Follow protocol instructions to prepare and add predesigned custom probe panels to the deparaffinized and decrosslinked slides and incubate Overnight .
Note
Tip: Make sure to record custom panel ID and slide ID as it will be used later for setup of the Xenium Analyzer.
8h
Post hybridization wash.
Note
Tip: Ensure the slide is not cracked and do not let them dry out. Change solutions one at a time. Do not let the slide cool, immediately add wash buffer after removing the hybridization solution.
Ligation and amplification. Add reagents for ligation, wash and then proceed to rolling circle amplification and wash.
Morphological marker staining for cell segmentation.
- After series of ethanol washing, incubate with xenium blocking and staining buffer.
- Prepare multi tissue staining mix that harbors cell segmentation staining reagents for cell nuclei, membrane and cytoplasmic boundaries and immune cells for morphology based segmentation plus protein marker panels (can choose all or a subset provided by 10X).
NOTE: follow protocol. Use the right kit if protein panel addition is desired. The protein markers can also be skipped, they do not affect the gene expression profiles in our experience on human kidney tissue.
- Incubate with staining reagents Overnight at 4 °C .
- After washes, treat with stain enhancer for 00:20:00 at Room temperature (start thawing autofluorescence quencher for next step).
8h 20m
Autofluorescence quenching. 00:10:00 at Room temperature in dark after washes and then post quenching wash in ethanol (normal light) and then in PBS-T in dark as in the protocol.
Note
Break point: can store the slides overnight in dark at 4 °C .
10m
Nuclei staining. Use nuclei stain reagents provided in the kit.
- after washes incubate with nuclear stain in the dark for 00:01:00 at Room temperature .
- Washes
Note
Brake point: can store slides at 4 °C in dark for less than a week.
Note
QC: check for tissue detachment and folding. These areas will not have signal and regions of fold may be oversaturated. Depending on extent of detachment or folding may determine if usable data will be generated. If the detachment area is less than 25% may proceed, but exceeding 70 % may not be usable. For intermediate may need to manually assess if there are still usable areas. These can be better assessed in the scan view on the analyzer in the next step.
1m
Imaging on Xenium Analyzer. Here we follow the protocol recommended by 10X chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://cdn.10xgenomics.com/image/upload/v1731971531/support-documents/CG000584_Xenium_Analyzer_UserGuide_RevG_update.pdf
Note
Ensure the correct modes are selected for the kit being used (v1 or prime). Also the kit if it includes protein marker panels.
Make sure the file for custom panel or other panel used are uploaded on the instrument and selected during run setup.
Perform readiness test to verify all systems are working and the instrument is ready for use.
Sample scan.
- After scan is complete, assess the scanned image for adequacy of staining as stated above in B6 above. Toggle different channels to ensure morphological markers have stained as expected.
Note
QC: Check for debris and detachments; if there is significant debris causing blurry scan do not proceed.
- Make sure to select at least 1 field of view (FOV) containing tissue. For multiple samples, specify each sample as separate region.
Transfer data after run is complete.
Xenium Data Quality. There are several levels of checking the quality of the Xenium in situ experiment. Some of these features should be directly visualized on the Xenium Explorer that can be freely downloaded from 10X https://www.10xgenomics.com/support/software/xenium-explorer/latest
- The initial QC tutorial is here https://www.10xgenomics.com/support/software/xenium-explorer/latest/tutorials/xe-checking-xenium-data-quality
Imaging quality. Check DAPI staining to assess tears, folds or detachment (QC).
Segmentation. Check quality of segmentation by examining cell segmentation polygon boundaries and nuclei. The cells contributed by each of the morphological marks can be seen. No segmentation method is perfect, if there are cells that are not segmented these can be exported and evaluated for their identity. Overcrowded areas such immune dense areas may have overlapping cells nuclei and may not segment accurately. Similarly if some cells are not desired to be analyzed, they can be selected using the lasso tool and exported, and these IDs can be removed, for example blood cells may not be desired, or non kidney tissue cells etc. For multimodal segmentation, 5um is used for the DAPI channel. Check if interior segmented cells have RNA stain and nuclei and the boundary segmented cells have boundary stain. Several cell types can be spot checked. Can also manually count select areas and compare with Xenium multisegmentation.
Cell clusters and appropriate region quality assurance. Can use Xenium default clustering or import own file and check on the cluster colors on the tissue to select cells and assess if the marker genes identify the cell type selected expected from the region. For example, in the kidney glomerulus check for parietal epithelial cells, podocytes, glomerular capillaries or adjacent macula densa or renin expressing cells.
Transcript quality. For select gene check Q score >20. Check different cell type marker genes to assess separation. Density map of all gene transcripts should show uniform coverage of the tissue.
Assay performance. Check for reproducibility if same sections are repeated and measure count of cells identified, transcript per cell and count correlation of genes among the two samples.
Analytical pipelines. Additional QC for rigor and reproducibility can be performed using custom analytical pipelines.
We use a combination of SquidPy, TACCO and scVI for pre and post QC analysis, integration, batch correction and clustering. A variety of other tools are used for neighborhood analysis including SquidPy, Banksy, MonkeyBread and NiCo. For cell annotation, KPMP-HuBMAP kidney atlas is used at different levels of annotation. We usually avoid using the degenerative tubular states for initial annotations as they show mismapping due to reduced expression of mature markers and common injury markers. These can be individually checked after subsetting the main clusters.
Pre-processing:
- Starts with Instrument Output, converted into Initial Data and then Flat Files.
- Tools like Squidpy are used to ensure data quality.
Quality Control:
- Identifies negative probes and calculates False Discovery Rate (FDR) and other QC metrics. FDR <1%
- Filters cells and genes to create a Final QC Object. Remove cells with Genes < 5 count. Remove genes not expressed in any cell. can also use a transcript count QC filter - can start conservatively with a cut off of < 5 transcripts per cell. One can track assay performance by using mean transcripts per cell as a metric for RNA and mean intensity per cell for protein. However this will vary depending on the number of custom panels for RNA (300 or 480 or aother combination) or protein (4 vs 27 or more)
Dimensionality Reduction and Integration:
- Natural log Normalization, PCA, neighbors, highly variable genes and UMAP clustering are performed using Squidpy (Theis Lab).
- scVI used for integration or batch correction if the goal is to perform global analysis.
- Annotation is supported by organ-specific atlases (e.g., TACCO from Broad Institute). This can be done at level 1 with main cell types or deeper levels 2 and 3 for more granular subtypes.
Knowledge-Based Downstream Analysis (can be done at specific region of a sample, whole sample of an individual sample or a group of samples)
- Focuses on:
- Spatial Clustering to identify niches.
- Neighborhood Analysis to assess cell interactions. Neighboranalysis in spatially resolved ST will depend on parameters chosen such as distance and this may depend on biological goals of how expansive microenvironment is desired. When more granular cell type levels are chosen, due to overlap in closely related cells, one may see cell neighbors that may not be physically plausible based on their regional location in the tissue. These should be easy to point to detect in the output and taken into account.
- Discovery of Spatially Variable Genes.
- Tools like Squidpy, Monkeybread, NiCo, GraphCompass, and BANKSY support this step.
Post Xenium H&E staining. Follow Xenium instructions to remove the cassette and reagents and perform routine H&E staining.
Remove Xenium Cassette
H&E Staining
- Milli-Q water – 00:02:00
- Hematoxylin – 00:03:30
- Rinse until clear with running water
- 0.5% HCl/70% EtOH (differentiation step) – 10-15 dips
- Rinse with running water
- 1% Ammonia water (bluing step) – 10-15 dips
- Rinse with running water
- 70% Ethanol – 20 dips
- Eosin (Add ~2 mL acetic acid to new eosin after it has been changed) – 00:00:10
- 95% Ethanol – 20 dips
- 95% Ethanol – 20 dips
- 100% Ethanol – 00:00:30
- 100% Ethanol – 00:00:30
- Xylene – 20 dips
- Xylene – 20 dips
- Xylene – store while cover slipping
Note: A quenching step can also be used to improve H&E staining. If another assay is desired on the same slide, then can wait for H&E staining.
6m 40s
Coverslip
Acknowledgements
Kidney Translational Research Center (KTRC)