Please refer to NSTG up-to-date morphology marker list in the following link: https://nanostring.com/support-documents/morphology-marker-clone-list/

Please refer to our up-to-date morphology marker list in the following link: https://docs.google.com/spreadsheets/d/1xhqf1zJHcA4fAj0LoC0lRqEC2fkk8U4HiFI39kkpv70

• RNAscope and Immunofluorescence staining are current standard for validating spatial findings.
• CosMx could also validate GeoMx findings however it is costly compared to prior two technologies.

• For RNA application (WTA), we would need around 150-200 cells minimum.
• For protein application, 20 cells minimum would be enough.
• It is recommended to have more if possible, but clean selection is encouraged over number of cells.

• Any positively charged slides would work, below is the slides we have used before;
  • VWR Superfrost Plus Micro Slide (Cat#: 48311-703)
  • Fisherbrand Superfrost Plus (Cat#: 12-550-15)
  • Leica X-tra Adhesives (Cat#: 3800050) slide
  • Matsunami PLATINUM PRO Adhesive Glass Slide
• If you use uncharged glass, LSG will not be responsible for tissue falling off during antigen retrieval.

• To calculate p-value, we would need 3 ROI or AOI per group.
  • For example, in a single slide with tumor sample that has clear tumor boundary and stained with morphology markers, SYTO13/PanCK/CD45/CD68:
  • Tumor versus Stroma versus Boundary could be compared
  • If comparison is done for each morphology marker;

In total 3(morphology markers)X3(region)X3(for p-value calculation); 27 AOI would be required. However, downside of this collection is lack of biological replicate.

• Nanostring GeoMx DSP have 4 channels, however, one of them is reserved for nuclear staining using either SYTO13 or SYTO83
• Remaining 3 channels can be determined based on sample and hypothesis.
• If there is no need for 3rd antibody, background staining using PanCK or Vimentin or a-SMA is recommended.

• Around 1 week for test staining (if opted)
• 1 week for GeoMx DSP run and library preparation
• 3 weeks for sequencing
• 1 week for analysis

• 2 slides for GeoMx DSP
• 2 slides for IF
• 2 slides for DV200
• 1 slide for H&E
• Number of slide depends on number of services selected.

• Best if it is within 2 weeks of GeoMx DSP run. We have run sections 3 months old, and it was acceptable.
• If sections are more than 6 months old, it is recommended to check RNA quality using RNAscope.
• If protein NGS assay will be used, old section would work stably.

• FFPE and Fresh Frozen Samples could be used.
  • For FFPE, you can submit your blocks and we can section it for you. If you are submitting slides, it is required to use positively charged slides to prevent tissue fall off during antigen retrieval. 5mm thickness is suggested on average.
  • While you can submit FFPE blocks older than 3 years, it is important to section within 2 weeks to 3 months of your GeoMx DSP run.
  • We would need 2 sections for GeoMx DSP, one for the actual run and one as a back up. If you would like to do mock ROI selection and test antibody staining, additional sections would be required.