Question: Is healthy skin compatible with the Visium CytAssist Spatial Gene Expression for FFPE assay?
Answer: Based upon in-house testing with H&E stained healthy mouse and human skin FFPE tissue sections we have determined they are a challenging tissue type for a few reasons we will describe here. Due to high lipid content and cross sectional surface area, skin sections are less sticky and are more prone to detachment from glass slides. In addition to detachment, a significant portion of reads from healthy skin tissues was attributed to genomic DNA (gDNA) lowering overall assay sensitivity (Genes and UMI counts per spot). Both of these factors may contribute to a Low Fraction Reads Mapped Confidently to Probe Set alert in the Visium Web Summary File. Due to these challenges, further optimization will be required for compatibility of healthy skin tissues with the Visium CytAssist Spatial Gene Expression for FFPE assay.
Outlined below is a summary of our learnings and considerations when working with healthy FFPE sections from human or mouse skin. Modifications to the Demonstrated Protocols and User Guide steps is currently unsupported and therefore is at the customer's own risk. 10x Support does not have additional information beyond what is provided in this article regarding processing healthy human or mouse skin FFPE tissue sections and will not be able to provide additional details or data from internal experiments.
1. Tissue Adherence: We commonly encountered tissue detachment when processing 5 µm healthy human skin sections on Superfrost Plus slides. Healthy mouse skin sections processed on Superfrost Plus slides did not experience tissue detachment.
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- We have determined 3 - 10 µm section thickness is compatible with FFPE tissues. Testing slightly thinner healthy skin sections (3-5 µm) may help promote tissue adhesion during the Visium CytAssist Spatial Gene Expression workflow.
- During limited testing with bone, skin, and human breast, we found that sample adhesion was significantly improved when 5 µm FFPE tissue sections were placed on specially coated hydrogel slides. Nexterion Slide H – 3D Hydrogel Coated (also available through Fisher) slides have not been fully validated but have performed consistently well with the limited number of samples and tissue types we have tested. Nexterion Hydrogel slides should be stored at -20°C per manufacturer’s recommendations, equilibrated to room temperature for 30 min before use. After tissue sections have been placed and dried, slides should be stored in a desiccator at room temperature until ready to process.
- Since healthy skin tissue is more prone to detachment, it is important to follow best practices when working with this tissue type. Ensure to follow recommended drying steps outlined in the Visium CytAssist Spatial Gene Expression for FFPE – Tissue Preparation Guide once sections are placed on compatible glass slides. During section placement, skin sections can expand quickly in the water bath and may benefit from a lower water bath temperature for floating. During coverslip addition and removal exercise caution to not disrupt tissue, avoid storage of slide with coverslip if possible as this can make coverslip removal more difficult. When slides are assembled in the Tissue Slide Cassette, dispense and remove reagents along the side of the well without touching or pipetting on top of the tissue section. Additional sample handling best practices are outlined here: What sample handling methods can be implemented to minimize the likelihood of FFPE tissue detachment for Visium CytAssist Spatial Gene Expression?
- Overfixation of FFPE tissue blocks can have an effect on tissue adhesion. If you have access to multiple blocks prepared with different fixation times, you may want to consider testing a section from each.
- Testing optimizations using Hydrogel Coated slides and/or optimizing section thickness and quality can be performed by assessing for tissue detachment during H&E staining, imaging, coverslip removal, and after Decrosslinking. These steps can be found in the following demonstrated protocol Visium CytAssist Spatial Gene Expression for FFPE – Deparaffinization, H&E Staining, Imaging & Decrosslinking.
2. Poor Gene Expression Data: Gene expression results from healthy human and mouse skin samples demonstrated poor mapping and low sensitivity.
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During the Decrosslinking step, crosslinked nucleic acids are reversed allowing for access of left hand side (LHS) and right hand side (RHS) probes during the downstream Probe Hybridization step. For healthy human skin FFPE sections, Decrosslinking at 95°C for 1 hour resulted in a large portion of reads attributed to gDNA indicating standard conditions with this tissue type may be too harsh. This can be minimized by performing decrosslinking at a lower temperature. However, Decrosslinking at a lower temperature impacts assay sensitivity, thus, additional modifications are required to the Decrosslinking and Probe Hybridization steps of the workflow to reduce UMIs from gDNA and improve assay sensitivity.
- Decrosslinking: Replace Decrosslinking buffer (PN-2000566) with TE buffer pH=9 from GeneMed (PN-10-0046). Perform Decrosslinking incubation at 70°C for 1 hour. TE Buffer does not require dilution and should be used at 1X.
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Probe Hybridization: Pre-Hybridization Mix requires addition of Tissue Removal Enzyme (PN-3000387) at a final dilution of 1:40,000. After Pre-Hybridization proceed directly to Probe Hybridization.
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Example for 11mm capture area (1 reaction without overage):
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Prepare 1:200 dilution of Tissue Removal Enzyme in EB Buffer.
- Add 199 µl of Buffer EB to a microcentrifuge strip tube
- Add 1 µl of Tissue Removal Enzyme.
- Pipette Mix 15x with pipette set to 150 µl and briefly spin down.
- Prepare Pre-Hybridization Mix using 1 µl of previously diluted Tissue Removal Enzyme into 200 µl final volume achieving a 1:40,000 final concentration.
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Prepare 1:200 dilution of Tissue Removal Enzyme in EB Buffer.
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Example for 11mm capture area (1 reaction without overage):
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During the Decrosslinking step, crosslinked nucleic acids are reversed allowing for access of left hand side (LHS) and right hand side (RHS) probes during the downstream Probe Hybridization step. For healthy human skin FFPE sections, Decrosslinking at 95°C for 1 hour resulted in a large portion of reads attributed to gDNA indicating standard conditions with this tissue type may be too harsh. This can be minimized by performing decrosslinking at a lower temperature. However, Decrosslinking at a lower temperature impacts assay sensitivity, thus, additional modifications are required to the Decrosslinking and Probe Hybridization steps of the workflow to reduce UMIs from gDNA and improve assay sensitivity.
The content of this article is specific to healthy human and mouse skin FFPE tissue sections. We have successfully processed diseased human skin (Melanoma) FFPE sections following the Visium CytAssist Spatial Gene Expression demonstrated protocols and user guide without any modification. We have not tested the modifications listed here with diseased human skin FFPE sections. It is important to consider your experimental design, as modifications to the processing of healthy versus diseased skin tissues will result in batch effects and therefore may confound analysis.
Product: Visium CytAssist for FFPE