Question: Do you have recommendations for sectioning TMAs for Xenium?
Answer: Tissue Microarrays (TMAs) are paraffin blocks that contain cylindrical cores of tissue. In general, TMAs are challenging to work with in terms of sectioning, placement, and risk of core-to-core cross-talk. TMAs are compatible with Xenium as long as the probe panel matches the tissue type. We have performed limited testing of TMAs internally and sectioning requires expertise. Generally speaking, the Xenium FFPE Tissue Preparation Guide and Xenium FFPE Deparaffinization/Decrosslinking Protocols have appropriate guidance for working with TMAs (e.g. the drying steps and deparaffinization steps will likely work just as well for TMAs as other FFPE tissues). While we always recommend following Demonstrated Protocols for Tissue Preparation on the Support Webpage, we have additional tips and tricks for working with TMAs for Xenium listed here.
Compatible TMA Features:
TMAs should fit within the 10.5 x 22.5 mm sample area on the Xenium slide, with an optimal 1 mm pitch between cores (edge to edge measurement; Figure 1). Core diameters <1 mm increases risk of detachment in the workflow. Cores with a diameter of 1 mm and a pitch of 0.5 mm can be used to maximize TMA placement the sample area, surveying up to 105 TMAs per Xenium slide (in a 7 x 15 core array).
Figure 1. Example images of sections generated from alternating cores of mouse lung and brain cores with 1 mm edge to edge distance between cores.
- Preparing TMA blocks is very challenging and we are unable to provide additional guidance on TMA block generation. We recommend using a service provider.
- Designing blocks with two different tissues in the same TMA presents some challenges:
- Different tissues may have different float times which can impact section placement.
- Two different base probe panels cannot be used on the same slide (e.g. mLung and mBrain). Thus, running a block such as the one shown in Figure 1 would require a custom panel design or compatibility with an upcoming human multi tissue panel.
- If the expression of genes in tissue is not matched with the panel chosen, there may be low quality scores due to low expression or imaging artifacts from overexpression..
- We have worked with Acepix Biosciences (Hayward, CA) and Novus Biologicals (Centennial, CO) to generate TMA blocks.
- If you have TMA blocks where cores tend to pop out of sections, it is likely that the cores in these blocks are not properly set. You could try warming the surface of the block to loosen the wax around the cores to help reset the cores prior to cutting. This would involve placing the block face down on a prewarmed glass slide (prewarmed to 40-43°C) for about 10 minutes. Then transfer the slide to an ice bath for rehydration prior to sectioning.
- We recommend changing the microtome blade in between block trimming and sectioning. Since TMAs tend to be on the wider side, using a new area of the same blade for sectioning is usually not as effective.
- Sometimes cores appear to have popped out of a section during cutting, but may actually just be curled. Oftentimes these curls will flatten out during the floating step. Try transferring "imperfect" sections to the water bath to check if this is the case to avoid exhausting the TMA block in an attempt to achieve the "perfect" section prior to floating.
- While we recommend ~42°C for most FFPE tissues, a low temperature water bath of ~37°C is usually better for TMAs. The temperature may need to be optimized for each block depending on the tissue embedded.
The standard guidance in the Xenium FFPE Tissue Preparation Guide is applicable to TMAs. However, if detachment is observed, TMAs may benefit from extended drying times.
- After sectioning, gently flick the slide to remove excess water and place the slide in a drying rack. Place slides at room temperature in a drying rack overnight (with no fan or desiccator). In the morning, perform the 42°C drying step for 3 hrs. Slides can be either placed in a desiccator for storage (according to Xenium FFPE Tissue Preparation Guide) or proceed to 60°C bake step for 2 hrs as outlined in Xenium FFPE Deparaffinization/Decrosslinking Protocols
- Follow the instructions in the Xenium FFPE Deparaffinization/Decrosslinking Protocol. Slow immersion during deparaffinization steps (especially the first immersion in xylene and subsequent ethanol steps) is critical for minimizing detachment.
- TMA core quality will impact assay performance. The H&E QC described in the Xenium FFPE Tissue Preparation Guide can be used to predict section adhesion. Poor or inconsistent staining may indicate future detachment.
Overall conclusions: While TMAs are tricky to work with, it is possible to obtain good Xenium data from cores.
10x Genomics Support does not have additional information on generating TMA blocks, sectioning, and placing sections beyond what is provided in this article and will not be able to provide additional details from internal experiments or guidance on troubleshooting TMA experiments at this time.
Products: Xenium In Situ Gene Expression