Question: How should I fix and embed my tissue for Visium for FFPE?
Answer: If starting out with fresh tissue, we would advise proceeding with our standard Visium assay (for snap-frozen and OCT embedded tissue).
Fixing and embedding is a nuanced process that is variable depending on tissue types. If using fresh tissue isn’t an option and the tissue needs to be fixed, we recommend the following fixation and embedding best practices which include, but aren't limited to:
- Ischemia time should be minimized, tissue should be fixed as soon as possible, and fixation and embedding conditions optimized. This will help to maintain tissue structure while minimizing autolysis and degradation of macromolecules (such as RNA).
- Tissue should be fixed in fresh cross-linking formaldehyde solution (such as 10% neutral buffered formalin (NBF) or 4% paraformaldehyde (PFA)) and then embedded in paraffin wax.
- General recommendations and best practices can be found in this Leica article: An Introduction to Specimen Processing.
As crosslinking fixatives can damage RNA and RNA fragmentation level impacts assay performance, the RNA quality of the FFPE blocks should be assessed prior to the workflow. If the FFPE blocks have high-quality RNA (DV200 ≥50%) these blocks may be compatible with the assay. Information on RNA Quality Assessment will be available in our Visium Spatial Gene Expression for FFPE Tissue Preparation Guide.
See these related Q&A articles:
- What fixation and embedding methods are compatible with Visium for FFPE?
- What is the difference between formaldehyde, formalin, and paraformaldehyde?
- How does over-fixation impact Visium for FFPE assay performance?
Products: Visium for FFPE