Where are the validated stopping points in the Visium CytAssist Spatial Gene Expression for Fixed Frozen assay?

Question: Where are the validated stopping points in the Visium CytAssist Spatial Gene Expression for Fixed Frozen (FxF) workflow?

Answer: Below are a list of validated stopping points during tissue preparation and library construction:

• During Tissue Preparation:
  1. Tissue blocks PFA fixed, sucrose cryopreserved, simultaneously snap frozen and OCT embedded, can be stored in a sealed container at -80°C for long term storage.
  2. Tissue Slides with fixed frozen OCT embedded tissue sections placed can be stored in a sealed container at -80°C for up to 2 months.
• During the Visium CytAssist Spatial Gene Expression for FxF assay: 
  • The workflow occurs over two days once Tissue Slides have been prepared. The first day includes rehydration, H&E staining, imaging, destaining, and overnight probe hybridization. The second day includes completing the on-slide RNA Templated Ligation (RTL) steps, use of the Visium CytAssist instrument to transfer analytes from two Tissue Slides to a Visium Gene Expression slide, followed by off-slide library construction steps.  
    • If done continuously, the first day is estimated to take ~2 hours (not including imaging time needed), and the second day is estimated to take ~7 hours.
  • Multiple stopping points have been built into both days of the workflow to maximize flexibility. Please refer to the Visium CytAssist demonstrated protocols and user guide for specifics regarding storage conditions at each of these stopping points.
    1. After H&E staining one of 2 optional stopping points can be selected. We have not validated combining these stopping points. Therefore, utilizing only one is recommended.

       1. After completing H&E Staining and coverslip mounting, slides can be stored laying flat in a container at 4°C, in the dark for up to 2 weeks. Coverslip application is required prior to storage.

       2. After completing coverslip application & imaging, slides can be stored laying flat in a container at 4°C, in the dark for up to 2 weeks. Coverslip application is required prior to storage.
    2. After completing Post-Ligation Washes, slides can be stored at 4°C for ≤24h.
    3. After completing Probe Extension, slides can be stored at 4°C for ≤24h.
    4. After completing Pre-Amplification SPRIselect Cleanup, samples can be stored at 4°C for ≤72h or at -20°C for ≤4 weeks.
    5. After completing Sample Index PCR and SPRIselect Cleanup, samples can be stored at -20°C for long-term storage.

Answer: Visium CytAssist for FxF