How can Xenium slides be stored prior to a Xenium Analyzer run?

Question: How can Xenium slides be stored prior to a Xenium Analyzer run?

Answer: Xenium slides can be stored short term (up to 1 week) or long term (4 four weeks* or 2 months**) after performing nuclei staining as indicated in the following protocols:

• Xenium In Situ Gene Expression User Guide (CG000582)**
• Xenium In Situ Gene Expression with Cell Segmentation Staining User Guide (CG000749)**
• Xenium Prime In Situ Gene Expression with optional Cell Segmentation Staining User Guide (CG000760)*

Short-term Storage (up to 1 week)

• Store covered slides in 1,000 μl PBS-T at 4°C in the dark
• Ensure that the slide is stored in microbe-free and nuclease-free conditions, with a Xenium Cassette Lid or slide seal applied to prevent evaporation
• If storage beyond 1 week is needed, we recommend transferring the slides to long-term storage

Long-term Storage (1 week+)

• Prepare cryoprotectant: 30% glycerol solution in PBS (RNase- and nuclease-free)
  • ! Ensure glycerol is diluted in PBS not water
  • ! Ensure media is thoroughly mixed
• Remove all PBS-T from the cassette well
• Add 1,000 μl 70% ethanol and incubate for 2 min at room temperature
• Add 1,000 μl 100% ethanol and incubate for 2 min at room temperature (1st incubation)
• Add 1,000 μl 100% ethanol and incubate for 2 min at room temperature (2nd incubation)
• Remove slide from cassette and place in a slide mailer containing sufficient cryoprotectant to fully submerge the slide. Do not discard slide cassette.
• Store slides in cryoprotectant at -20°C for up to recommended storage time
• Clean slide cassettes and store until needed
  • Rinse under running Milli-Q water
  • Spray with 70% isopropanol
  • Repeat Milli-Q water and 70% isopropanol wash
  • Rinse under running Milli-Q water
  • Air dry

During storage, the slide is likely to freeze within the 30% glycerol media. Here we see examples of Xenium slides stored in slide mailer (Figure 1) and a conical tube (Figure 2).

Figure 1. Pink slide mailer with a single xenium slide surmerged in 10mL of cyroprotectant.

Figure 2. A single Xenium slide in a connical tube filled with cryoprotectant media.

Using Slides Stored at -20°C

• Equilibrate slide mailer to room temperature
  • ! It is important to thaw completely to ensure no ice crystals remain during slide removal as these could scratch the slide
• Remove glycerol and rinse slide mailer 3x with PBS-T (10 ml or volume sufficient to submerge slides)
• Remove slide from mailer and assemble into stored slide cassette
• Add 1,000 μl PBS-T to the cassette well

Slides can be stored after Xenium Analyzer run (or in the event of a run failure) in a similar manner. How does slide storage or rerunning slides on the Xenium Analyzer impact data. Please reach out to support@10xgenomics.com for more information.

If post-Xenium run outputs are desired (e.g. CytAssist or IF), proceed immediately with IF or CytAssist for optimal assay performance (documentation here). Only perform short term storage in PBS-T and proceed to post-run application within 3 days.

Products: Xenium In Situ Gene Expression, Xenium Prime In Situ Gene Expression, Xenium Analyzer