Question: What should I do if I cover fiducials on the Xenium slide?
Answer:
It is critical that fiducials are not covered during tissue placement, as these could result in instrument errors. If a mistake occurs, 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 tissue adhesion (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 (support@10xgenomics.com) for assistance before proceeding with the Xenium workflow and instrument run.
Support cannot provide replacements for reagents lost or poor assay performance due covering fiducials or any mitigation strategies presented here.
Minimum Fiducial Requirements (all of the following must be met):
- Top: 2
- Bottom: 2
- Top + Bottom: 6
- Center: 3
- Total: 9
Covering Fiducials
The Xenium Analyzer needs to detect a minimum number of fiducials before a run can initiate. Tissue, glue, marker, or any reagent that changes the appearance of the fiducials can lead to instrument errors. If fiducials are hard to visualize, use a cell phone camera with the slide on a black background to aid in visual inspection of the slide.
If fiducials are accidentally covered, we recommend purchasing additional slides and repeat tissue placement. If it is not possible to repeat sectioning, it may be possible to proceed with the original sample, though 10x Genomics will not support or provide replacement reagents for tissue detachment or instrument fiducial errors.
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 liquid from the cassette well.
- Gently unmount slide from cassette. Instructions can be found in the Xenium Quick Reference Card for Slide Cassette Assembly.
- 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 Xenium Quick Reference Card for Slide Cassette Assembly.
- 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.
Summary
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