Apr 28, 2026
Version 2
Identification of optimal pre-treatment by Xenium Tissue Optimisation (XTO) V.2
- 1Wellcome Sanger Institute
- Human Cell Atlas Method Development Community
Protocol Citation: Kenny Roberts 2026. Identification of optimal pre-treatment by Xenium Tissue Optimisation (XTO). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldr819g5b/v2Version created by Kenny Roberts
Manuscript citation:
Optimisation of Xenium automated in situ sequencing for PAXgene-fixed tissue samples
Roberts, K. and Bassett, A.R.
Journal of Histotechnology, 2026
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 have used this protocol successfully for various PFPE human tissues.
Created: April 18, 2026
Last Modified: April 28, 2026
Protocol Integer ID: 315292
Keywords: in situ sequencing, Xenium, PAXgene, PFPE, tissue, optimisation, smFISH, ISS, permeabilisation, 10x Genomics, xenium tissue optimisation, tissues for xenium, xenium, replicate tissue section, optimal conditions for transcript detection, efficient optimisation of pretreatment, preservation of tissue morphology, transcript detection, different permeabilisation conditions in order, tissue section, tissue morphology
Funders Acknowledgements:
Wellcome
Grant ID: 220540/Z/20/A
Abstract
The Xenium Tissue Optimisation (XTO) protocol allows efficient optimisation of pretreatment of tissues for Xenium automated in situ sequencing. By utilising a multi-well gasket architecture, replicate tissue sections may be treated with different permeabilisation conditions in order to find the optimal conditions for transcript detection and preservation of tissue morphology.
Guidelines
For further information about the development of this protocol, and the interpretation of the resulting data, see the associated manuscript: Optimisation of Xenium automated in situ sequencing for PAXgene-fixed tissue samples, Roberts and Bassett, Journal of Histotechnology, 2026, http://dx.doi.org/10.1080/01478885.2026.2667003.
For cost-efficiency, knowing that only a fraction of the slide will generate optimal data, it is recommended to use Xenium v1, with either a pre-designed panel (Multi-Tissue, Brain, Immuno-Oncology) or a custom panel suited to the tissue of interest, for the XTO protocol itself. Once optimal conditions have been identified, those same conditions may be used upstream of the Xenium Prime 5000-plex workflow too.
It is important to use the same pepsin for the XTO protocol and any subsequent experiments. Different sources of pepsin may have different activity levels (both advertised and actual) and these may not behave similarly. For example, Sigma P7012 (≥2,500 units/mg) is a more concentrated form of the enzyme than Sigma P7000 (≥250 units/mg) and is provided as lyophilised flakes that are extremely static-prone and more difficult to dissolve thoroughly. The higher activity per unit mass makes the product more prone to autolysis and we have anecdotally found that activities of resulting solutions of P7012 are prone to be: a) less consistent; and b) not proportional in activity to solutions made with P7000.
Materials
General consumables
Nuclease-free water AmbionCatalog #AM9932
10xPBSAmbionCatalog #AM9624
Pepsin from porcine gastric mucosaMerck MilliporeSigma (Sigma-Aldrich)Catalog #P7000
Tween™ 20 Surfact-Amps™ Detergent SolutionThermo FisherCatalog #28320
10x Genomics Visium consumables
Visium Accessory Kit10x GenomicsCatalog #1000194
Visium Spatial Tissue Optimization Slide & Reagent Kit10x GenomicsCatalog #1000193
10x Genomics Xenium consumables
Xenium Slides & Sample Prep Reagents10x GenomicsCatalog #1000460
Xenium Decoding Reagents10x GenomicsCatalog #1000461
Xenium Decoding Consumables10x GenomicsCatalog #1000487
Optional, recommended:
Xenium Cell Segmentation Add-on Kit10x GenomicsCatalog #1000662
Safety warnings
Unscreened human tissues may be host to aventitious biological agents including blood-borne pathogens, such as HIV, hepatitis B, hepatitis C, and malaria. Fixation with PAXgene and subsequent processing to paraffin is expected to deactivate many pathogens but consideration of any particular pathogens must be made when assessing the appropriate biosafety containment level of the laboratory in which this protocol is performed.
This protocol may generate solid biological waste (tissue), biologically-contaminated sharps waste (blades, glass slides), and chemically contaminated liquid waste (solvents). All should be handled and disposed of in accordance with local regulations.
This protocol requires several hazardous chemicals including xylene (toxic, damaging to organs), ethanol (highly flammable), and pepsin (irritant). Xylene must be used only in ventilated infrastructure such as a ducted fume cabinet.
For full text of hazard statements, check relevant Materials Safety Datasheets.
Sharp tools, including microtome blades and scalpels, are required in this protocol. Use with care (including disposal), and take note of any local guidelines for biologically contaminated sharps injuries.
Ethics statement
Handling, use, storage, and disposal of human tissues must be conducted in a respectful manner in line with Human Tissue Authority (HTA) guidelines, reflecting the sensitive nature and origin of the material.
Ensure prior to start that collection, carriage, and receipt of tissues is compliant with all HTA guidelines, including Research Ethics Service/Committee approval, and thorough labelling and tracking of all human materials.
Before start
Before starting this protocol:
- It is important to select a sample(s) that will be representative of the cohort to be analysed but also compatible with the restrictive sectioning regime needed. The smallest XTO capture area is approximately 4 x 4 mm.
- Ensure that you are comfortable with the sectioning required - placing up to six paraffin sections in such close proximity is challenging.
- Read the protocol thoroughly, as there are various parallel steps with time sensitivity.
- It is recommended to practice assembly and disassembly of the Visium TO cassette using a blank Superfrost slide as this is a delicate procedure that can put the slide under a lot of strain and if done incorrectly it is likely to result in the slide breaking and being unusable.
Sectioning
Use the XTO sectioning template to divide a Xenium slide into four or six sample areas, depending upon the size of the tissue to be used. Use an ethanol-soluble marker pen such as a STAEDTLER Permanent Pen to trace the capture areas on the back of the Xenium slide. Take care to not mark the front (sample side) of the Xenium slide.
The layout of the XTO: overlaying the sample capture areas of the Xenium slide and the Visium TO slide yields six small capture areas (which may be combined into four if the smallest of the six are too small for meaningful tissue representation).
It is recommended to also mock up some Superfrost slides with the same XTO layout drawn on the back, in order to practice placement of sections. Removal of sections from a Xenium slide is not recommended and so it is essential to be comfortable with the challenging nature of sectioning required prior to placing sections onto the XTO slide.
Prepare samples and equipment for sectioning as per normal Xenium sectioning using a microtome.
Face the PFPE blocks by removing excess wax and the first few microns of tissue and hydrate on ice, pre-heat a water bath to ~45ºC or whichever temperature is optimal for your samples. Chill forceps on ice.
General advice may be found in the routine Xenium protocol for FFPE tissue (C000578 Xenium In Situ for FFPE – Tissue Preparation Guide).
Collect several ribbons of sections (recommended thickness 5 μm as per normal Xenium) and float onto a water bath.
You may wish to collect a section or two onto Superfrost slides for later H&E or other staining.
A section placed onto a Superfrost slide may also be useful for 'sizing up' against the XTO sample capture areas.
Using chilled forceps, separate sections from the ribbon, and remove any excess wax around the section. Depending upon the cross-sectional area of the tissue block and any regions of particular interest or anatomical representation, it may be necessary to trim sections to remove some tissue as well as wax.
It is critical that sections do not fall outside the XTO compatible sample areas; any tissue overlapping the gasket areas will not be informative and it is crucial that samples do not overlap one another.
It is recommended to use the mock slides prepared in step 1 to practice placement. Placing as many as six sections onto a slide in such close proximity is challenging and it may be necessary to experiment with holding and immersing the slide in atypical ways and angles in order to avoid previous sections detaching from the slide.
Continue until all four or six sections are in place.
Allow the slide to air dry at room temperature (optionally with a fan blowing air gently) in order to remove visible water from the slide.
Examples of sectioned XTO slides: left, six sections of PFPE mouse cerebellum; right, four sections of mouse kidney. Blue marker pen lines of the back of the slide will be removed prior to Xenium processing.
Once the slide is visibly dry, bake at 42ºC for 3 hours as per the recommended 10x Genomics protocol for FFPE sections for Xenium (C000578 Xenium In Situ for FFPE – Tissue Preparation Guide).
Once slides have been dried and baked, store for up to 1 month in a slide mailer or small slide box in a dessicator under vacuum and/or with dessicant beads.
This is based upon the recommended 10x Genomics protocol for FFPE sections for Xenium (C000578 Xenium In Situ for FFPE – Tissue Preparation Guide). It is possible that prolonged storage is acceptable but this has not been tested, particularly for PFPE sections, which may be less stable than FFPE.
De-paraffinisation and preparation for XTO
Place the Xenium v1 thermocycler adaptor into the thermal cycler and pre-heat to 60ºC.
Bake the slide(s) at 60ºC for 30 minutes.
Use a tissue moistened with 70-100% ethanol to remove any pen marks by gently wiping the back of the slide before or after baking.
During the 30 minute bake step, prepare the reagents needed for de-paraffinisation, as per the 10x Genomics protocol for FFPE pre-treatment (CG000580 Xenium In Situ for FFPE – Deparaffinization & Decrosslinking).
Briefly: 2 x 100% xylene; 2 x 100% ethanol; 2 x 70% ethanol; 1 x nuclease-free water.
It is recommended to prepare 40 ml of each solution in a 50 ml Falcon tube.
Also prepare ~500 ml of 1x PBS.
After 30 minutes, remove the slide(s) from the thermocycler adaptor and equilibrate to room temperature for a few minutes.
Start de-paraffinisation: incubate the slides in 100% xylene for 2 x 10 minutes. Use forceps to hold and transfer the slides, taking care to hold only by the label area and avoid making contact with the sample area or tissue sections.
During xylene incubations prepare pepsin, a heat block, and a thermocycler:
Carefully transfer approximately 20-50 mg of pepsin into a 1.5 ml tube or the lid of a 50 ml tube.
Tare a fine balance with an empty 50 ml Falcon tube, and then carefully add pepsin powder from the aforementioned aliquot with a fine spatula, noting the exact mass added. Discard the rest, and seal the 50 ml tube with a clean lid.
Pepsin powder may be extremely light and prone to static. It is recommended to remove static from tubes, etc. using an anti-static gun. N.B. targeting the balance itself may disrupt accurate measurement.
N.B. note that there are multiple pepsin products available from Merck (Sigma) and other suppliers and each may have different activity levels. This protocol is designed for Merck P7000, which has an activity of ≥250 U/mg. Other activity levels may require different dilution.
Place the Visium thermocycler adaptor into the thermal cycler and pre-heat to 37ºC.
Pre-heat a heat block suitable for 1.5-1.7 ml microcentrifuge tubes to 37ºC.
Following xylene incubations, dehydrate slides through 100% ethanol and 70% ethanol, for 2 x 3 minutes each.
During ethanol incubations, dissolve and aliquot pepsin:
Add 0.1 M HCl to the pepsin in order to produce a 1 mg/ml solution. E.g. if the mass of pepsin was 21 mg, then add 21 ml of 0.1 M HCl.
Gently swirl and invert the tube to re-suspend and dissolve the pepsin. The solution may remain a little cloudy but should not have visible lumps or flakes.
Make several aliquots of 1.2-1.5 ml of the pepsin solution into 1.5-1.7 ml microcentrifuge tubes. Keep at room temperature for now.
During the final 70% ethanol incubation, place pepsin aliquots into the heat block at 37ºC. Take note of the time that incubation starts, and ensure that pepsin solution is pipetted onto the XTO slide within 15 minutes. Pre-heating of aliquots can be staggered to account for processing multiple time courses or slides.
After 70% ethanol, immerse the slide(s) in nuclease-free water and gently dunk up and down with forceps to rehydrate for 10-20 seconds.
Xenium Tissue Optimisation (XTO) permeabilisation
Remove excess water from slides with a quick flick and carefully wiping the back of the slide with a tissue.
Place the slide onto the Visium TO thermal cycler adaptor at 37ºC and dry uncovered for 2-3 minutes.
Remove the dry slide from the thermal cycler and assemble the Visium TO gasket and cassette.
Take care to assemble the cassette gently, using the Visium Slide Alignment Tool to prevent excessive force on the slide, which may result in breakage. Briefly: insert the grey cassette into the Slide Alignment Tool, which will hold it open; insert the rubber gasket and align with the cassette openings; place the Xenium slide into the assembly with the tissue sections facing the gasket, and press down to secure the slide underneath the clips. Carefully remove the assembly from the Slide Alignment Tool. A seal should be visible where the rubber gasket meets the Xenium slide.
Cut small pieces of self-adhesive seal to cover areas of the gasket. It is possible to use a single seal for the entire XTO but using smaller pieces to cover only 1-3 reaction chambers each reduces the risk of causing spills or splashes when removing each time to add pepsin.
It is helpful to note the time course on the top of the cassette, either with the duration of permeabilisation for each chamber or the time point at which pepsin should be added.
Place the XTO slide assembly on the Visium thermocycler adaptor on the thermal cycler at 37ºC and preheat for around 3-5 minutes.
During this time, ensure that the components for the next steps are at hand. It is recommended to situate the heat block with the pepsin as close to the thermal cycler as possible.
Fill a small box (such as a clean pipette tip box) with around 200 ml of 1x PBS for quenching after the time course.
Work quickly and carefully during the XTO time course. Use 200 µl of pepsin solution per chamber for the 6-chamber XTO configuation, or 300 µl per chamber for the 4-chamber XTO configuration.
Take the required volume of pepsin solution into a pipette, peel back the adhesive seal on the t=0 (i.e. the longest digestion time) chamber and add pepsin before immediately re-sealing it and closing the thermal cycler lid. Immediately start a timer.
Repeat the above step at each time point, adding pepsin to chambers one by one. Work quickly to minimise the time that the thermal cycler lid is open.
At the end of the time course, remove the XTO slide assembly from the thermal cycler and rapidly quench the permeabilisation by removing all of the self-adhesive seals and plunging the assembly into PBS.
Invert the assembly to drain liquid and then re-immerse in PBS to thoroughly wash the reaction chambers.
Remove the Xenium slide from the Visium TO cassette using the Visium Slide Alignment Tool to prevent excessive force on the slide, which may result in breakage. Immerse the slide in PBS for a final wash.
Xenium processing
Once slides have been rinsed and removed from the Visium TO cassettes, they may be processed according to the usual Xenium protocol, beginning with assembly into Xenium cassettes and then:
- Probe hybridisation for Xenium v1 (480-plex) (C000749 Xenium In Situ Gene Expression with Cell Segmentation Staining).
OR
- Priming oligo hybridisation for Xenium Prime (5000-plex) (CG000760 Xenium Prime In Situ Gene Expression with optional Cell Segmentation Staining).
The remainder of the Xenium protocol is unchanged, including running on the Xenium instrument (C000584 Xenium Analyzer).
Take care to reconcile the section positions with the permeabilisation conditions, noting that the Xenium Analyzer images samples with the label towards the user, which is reversed compared to typical slide handling (with the label facing away).
Protocol references
This protocol was used to optimise the application of Xenium to PAXgene-fixed paraffin-embedded human thyroid tissue in this publication: Nicola et al. Polyclonal selection of immune checkpoint mutations in thyroid autoimmunity, Nature 2026, https://www.nature.com/articles/s41586-026-10493-9; and in the associated data repository at the BioImage Archive https://www.ebi.ac.uk/biostudies/bioimages/studies/S-BIAD3103.