Question: What should I do if I cover fiducials on the Xenium slide?
Answer: It is very important not to cover fiducials during tissue placement, but mistakes do occur. In these instances, it is not possible to remove tissue sections and repeat sectioning on the same Xenium slide. This is because the Xenium slide chemistry is carefully optimized to minimize detachment and touching or wiping the slide will negatively affect the adhesive properties (See: Is it possible to reset a Xenium slide if tissue is placed incorrectly?). It may be possible to salvage slides and proceed with Xenium Analyzer run, but only under limited circumstances. The methods outlined below should only be used in troubleshooting; fiducials should not be covered as a common practice. If fiducials were accidentally covered during sample placement, please reach to Support (firstname.lastname@example.org) to evaluate slides, as needed, before proceeding with the Xenium workflow.
Support cannot provide replacements for reagents lost or poor assay performance due covering fiducials or any mitigation strategies presented here.
The Xenium system needs to see various fiducials and the coverage can be complex. The fiducials are laid out in a frame. Similar to Visium, one would not want to cover that frame. Multiple sections can be placed within the frame to maximize capture area. In any fiducial-based system, one would not receive data on or outside of the fiducials. In general, we advise to avoid covering these. Covering a certain number of fiducials may be permissible, though not recommended. Covering corner fiducials will lead to run failure. If fiducials are hard to identify by eye, use a cell phone camera to aid in visual inspection of the slide.
- At most 3 fiducial markers in the top two rows may be covered
- At most 3 fiducial markers in the bottom two rows may be covered
- At most 5 fiducial markers in the middle rows may be covered
It is critical to adhere to the rules listed above or the instrument run may fail to initiate. If fiducials are covered, determined by visual inspection of slides, we would recommend purchasing additional slides and repeat tissue placement (PN-1000465). If this is not possible to repeat sectioning, it may be possible to proceed with the original sample, although officially unsupported.
Slides would be processed with no modifications following the sample preparation and assay workflow (through nuclei staining). After nuclei staining, perform the following (at your own risk, as assay performance may be compromised):
- Remove any liquid in the cassette well.
- Gently unmount slide from cassette. Instructions can be found in the Assay User Guide ‘Cassette Removal Section’ (CG000582).
- Using a pipette tip, gently scrape off tissue on or outside of the fiducial frame. Be careful not to disturb tissue.
- Remount cassette. Instructions can be found in the Assay User Guide ‘Cassette Assembly Section’ (CG000582).
- Add 1,000 μl PBS-T.
- Proceed with Xenium run.
FFPE sections only
For FFPE sections that have been placed over fiducials and have not been dried yet, it is possible to submerge the slide again and gently refloat sections in a water bath. Placement can be repeated, ensuring that minimal water is trapped beneath the section and the section does not begin to disintegrate from floating too long.
It is critical to identify potential problems with fiducial covering prior to the Xenium Analyzer overview scan. Once the overview scan is initiated and a fiducial finding error is detected, the run must be aborted and the decoding reagents and consumables discarded.
It is also important not to wipe off tissue early in the assay workflow as this may compromise tissue integrity and lead to increased risk of detachment.
Product: Xenium In Situ Gene Expression