Question: How does delayed fixation impact Xenium performance?
Answer: Delays in fixation can lead to autolysis and degradation of RNA and proteins. Minimizing the time between sample collection and fixation is important for optimal Xenium assay performance. Below are observations made from a proof of concept experiment aimed at identifying the effects of delayed fixation on a sample’s molecular integrity.
Mouse brain tissue was harvested at four different time points.
- Control - immediate fixation
- 3 day - storage at 4°C in media/1x PBS
- 7 day - storage at 4°C in media/1x PBS
- 2 week - storage at 4°C in media/1x PBS
Tissue was then fixed according to our in-house protocol, utilizing tips and best practices outlined in:
- Do you have tips and tricks for pre-fixation tissue handling for Xenium?
- Do you have recommendations for FFPE tissue fixation for Xenium?
Mouse brains with different post-mortem fixation intervals were assessed for tissue integrity with H&E on standard histology slides.
The four images (A-D) illustrate coronal sections of mouse brains fixed at different time intervals post-harvesting: (A) Immediate fixation, serving as the control; (B) 3-day delay; (C) One-week delay; and (D) Two-week delay. Notable degradation in cellular integrity and staining uniformity can be observed as the time delay increases, highlighting the detrimental effects of delayed fixation on tissue morphology and histological interpretation.
We can then zoom in on the tissue and observe degraded nuclei after delay fixation. Not only is tissue morphology degraded, as shown above, nuclei shrink as well. Starting after 3 days delayed fixation, DNA condenses and shrinks.
Serial sections from the same four blocks / timepoints were run through the Xenium In Situ Gene Expression assay workflow followed by data generation on the Xenium Analyzer. Results are shown here:
Delayed fixation resulted in a decrease of the transcripts per cell detected, likely due to RNA degradation.
RNA density plots of all four timepoints show sheet decline in RNA density throughout the tissue.
It is important to minimize the time between tissue removal and fixation. Prolonged storage in cold reagents, such as 1x PBS or media, is not optimal for Xenium. Delayed fixation is a major driver for low transcript density. This said, multiple factors related to sample quality could contribute to low transcript density in an additive manner.
Product: Xenium In Situ Gene Expression