Jun 24, 2025

CosMx for FFPE V.2

DOI
https://dx.doi.org/10.17504/protocols.io.3byl41mrolo5/v2
  • 1Stanford University
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: [email protected]
Joanna Bi
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl41mrolo5/v2
Protocol Citation: Joanna Bi 2025. CosMx for FFPE. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl41mrolo5/v2Version created by Joanna Bi
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 24, 2025
Last Modified: June 24, 2025
Protocol  Integer ID: 220950
Keywords: cosmx for ffpe cosmx protocol, ffpe cosmx protocol, ffpe tissue, cosmx, ffpe, tissue
Abstract
CosMx protocol for FFPE tissue
FFPE blocks were sectioned into 5 µm sections using a microtome, placed onto BOND Plus Slides (Leica, SKU S21.2113.A), and dried as instructed prior to use (MAN-10159-02, NanoString). Sections were positioned within the imageable area as defined in the manual.
Dried sections were stored at 4°C until use.
Slides were baked at 60°C for 18 hours.
The FFPE sections were then deparaffinized and subjected to antigen retrieval at 100°C using Bond Epitope Retrieval Solution 2 (Leica, AR9640).
Incubation frames were assembled on the slides, and sections were permeabilized with Proteinase K at 40°C for 30 minutes, followed by a 5-minute fiducial incubation.
Post-fixation was carried out using 10% NFB, followed by Tris-Glycine treatment and blocking with NHS-acetate.
The sections were then incubated overnight with the CosMx Human Universal Cell Characterization Panel, which consists of a 950-target probe panel and 10 controls (NanoString, cat# 121500002).
After incubation, sections were washed to remove excess probes.
Cell membranes were then stained using morphology markers including CD298, PanCK, CD45, and CD68 from the CosMx Segmentation and Supplemental Markers Kit (NanoString, cat# 121500020, 121500021, 121500022).
Prepared slides were loaded onto the CosMx SMI instrument for imaging, following the NanoString manual (MAN-10161-02-1, Software Version 1.1). After pre-scanning each slide, regions of interest (ROIs) were selected as fields of view (FOVs) for further characterization.
The acquired scan data was then uploaded to NanoString’s cloud-based analysis platform, AtoMx, for downstream processing and analysis.