Trekker FX protocol videos
See how it works! These Trekker FX protocol videos demonstrate key steps in the Trekker FX workflow to generate spatially tagged single nuclei from formalin-fixed paraffin-embedded (FFPE) tissue samples.
Please note that these protocols only apply to Trekker FX assays for FFPE tissue.
Tissue section preparation
Learn how to properly section your FFPE tissue for Trekker FX spatial tagging.
TIPS
- Selection of section thickness is dependent on the desired nuclei yield and output. Thicker sections will generate higher nuclei yield following isolation and single-cell prep, but may impact H&E staining/imaging quality. Thinner sections will generate higher quality H&E staining/images, but may impact the nuclei yield and spatial coverage.
- If visible cracking or chattering of the tissue is observed, immediately STOP sectioning. The tissue section might be overly dehydrated. Remove the tissue block and rehydrate by incubating on wet ice for >15 min.
- The amount of time needed for the tissue section to flatten and dry will depend on the tissue type and condition of the block. Allowing too little or too much flattening time may result in wrinkled sections or breaking up of the tissue, respectively. Allowing too little or too much drying time may result in water being trapped underneath the section or the section detaching from the slide (see example times in Table 2 of the Trekker FX user manual).
Deparaffinization and decrosslinking
Learn how to deparaffinize and decrosslink your tissue section before spatial tagging with Trekker FX.
TIPS
- It is critical to trim the excess tissue from the Tissue slide prior to deparaffinization to maximize the yield of spatially tagged nuclei within the final preparation.
- When handling multiple samples or tissue types, use a new and clean single-edge razor blade for each sample to prevent potential cross contamination.
- Depending on the model of heat block or bead bath, it may take considerable time for the instrument and solution to warm up. Prepare the Decrosslinking Solution and incubate at 70°C for at least 30 min prior to the decrosslinking step.
Alignment fixture assembly
Learn how to assemble the alignment fixture, which ensures that your tissue is properly aligned with the Trekker FX tile.
TIPS
- The bottom plate of the Trekker Alignment Fixture allows for some rotational freedom for Tissue slide placement. Orient and install the Tissue slide in such a way as to maximize overlap with the Trekker FX Tile.
- When installing the double-sided adhesive, verify that there are no folds or creases and that both slides are flush against their respective plates. Distortions or differences in height may impact efficiency of spatial tagging or cause tissue detachment.
- If visible bubbles are present on the surface of the tissue or tile, pop using a pipette tip without touching the surface. Alternatively, gently wick and reapply the Cleavage Solution.
UV cleavage
Learn how to perform the UV cleavage step, which releases the spatial tags from the Trekker FX tile and enables spatial barcoding of individual nuclei.
TIPS
- If bubbles are present between the tissue and Trekker FX 10x10 Tile, DO NOT proceed with UV cleavage as this may lead to improper or inefficient spatial tagging. Disassemble and reassemble until no bubbles are present. Alternatively, gently wick and reapply the Cleavage Solution.
- If the Trekker FX 10x10 Tile and Tissue slide are not aligned, the fixture can be disassembled, and the slides repositioned to target the region of interest. Spatial tagging does not happen until UV cleavage occurs.
Nuclei isolation
Learn how to isolate your spatially tagged nuclei.
TIPS
- The total dissociation time will vary between tissue types. It is important to perform tissue optimization with the Trekker FX Training Kit to determine the ideal time for your sample. During optimization or when necessary, check the nuclei quality using a microscope after each trituration round. Failure to fully dissociate tissue or over-lysis may lead to reduced nuclei yield and loss of tissue regional representation.
- Depending on the tissue type and species, larger filter pore sizes (such as the pluriStrainer Mini 40 µm cell strainer) may be used to minimize filter clogging and maximize nuclei yield.
- DO NOT exceed 1 min of pestle grinding. Excessive pestle grinding or prolonged grinding can cause over-lysis and sample clumping, leading to reduced nuclei yield and compromised quality.
Nuclei stabilization
Learn how to prepare your nuclei for downstream single-cell analysis.
TIPS
- Counting with a fluorescent automated counter or microscope is strongly recommended. The use of Trypan Blue can lead to overestimated nuclei counts as the stain cannot differentiate between high-quality nuclei, poor-quality nuclei, and debris.
- Nuclei are stable for up to 5 hours on ice. Proceed to single-cell capture within the same day as isolation.
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