Question: How does over-fixation impact Visium for FFPE v1 assay performance?
Answer: This article provides an overview of Web Summary File metrics for Visium Spatial Gene Expression for FFPE v1 libraries from different tissue blocks across various fixation times. While DV200% may not accurately predict assay sensitivity with over-fixed samples, over-fixed samples generated similar spatial gene expression data as optimally-fixed samples. Individual results may vary depending on the particular sample type and quality.
In this experiment, mouse placentas were dissected and cut into pieces. A single piece from each organ was fixed for one day, ten days, fifteen days, or one month. When fixation was complete, tissues were processed and embedded to generate FFPE blocks. Several sections were taken from each block for RNA quality assessment and library preparation.
Table showing that extended fixation impacts assay performance. Lower DV200% values did not always correlate with longer fixation times but consistently lead to lower sensitivity as assayed by Genes and UMI Counts per Spot.
Spatial clustering (top) and t-SNE projections (bottom) of mouse placenta at one day, ten days, fifteen days, and one month (displayed from left to right) showing that longer fixation times and assay performance are negatively correlated.
As a best practice, tissues should be removed from NBF after fixation. We recommend to embed immediately following fixation for optimal results. If you are not ready to do so, the tissue can be rinsed with dH2O and stored in 70% ethanol at 4°C for up to one week until ready for paraffin-embedding. Keeping the tissue in alcohols/chloroform for longer may cause the tissue to slowly disintegrate or fall apart (especially for fattier tissues), which can impact sectioning. We would recommend checking the DV200 score to check for RNA degradation. Customers should perform a small pilot for over-fixed samples to determine how much the long fixation time will impact their data.
Products: Visium for FFPE