Seeker protocol videos
See how it works! These Seeker protocol videos demonstrate key steps in the Seeker workflow to produce sequencing-ready libraries from fresh-frozen tissues and obtain high-resolution spatial transcriptomic information.
Tissue melting on tile: Precise placement method
Sample is placed onto a tile with precise alignment. This method is recommended if the sample is significantly larger or smaller than the tile.
Tissue melting on tile: Stamping method
Sample is stamped onto a tile. This method is recommended if the sample is the same size as the tile and does not require precise alignment.
TIPS
- To flatten the sample after sectioning, use a brush to gently press the sample section into the cold block. Turning the sample section over can also reduce the curling of the OCT (optimal cutting temperature) compound.
- After melting the sample onto the tile, immediately proceed to process the sample or store for later use. DO NOT place the tile with the melted sample back on the cryostat. This will cause the sample to refreeze, which may reduce downstream data quality for some tissues.
- To store for later processing, freeze the slide with the tile and melted sample on dry ice, place it in a slide container, and store it at –80°C.
TIPS
- To flatten the sample after sectioning, use a brush to gently press the sample section into the cold block. Turning the sample section over can also reduce the curling of the OCT (optimal cutting temperature) compound.
- If needed after applying the stamping method, add a finger underneath the tile to further melt the sample onto the tile.
- After melting the sample onto the tile, immediately proceed to process the sample or store for later use. DO NOT place the tile with the melted sample back on the cryostat. This will cause the sample to refreeze, which may reduce downstream data quality for some tissues.
- To store for later processing, freeze the slide with the tile and melted sample on dry ice, place it in a slide container, and store it at –80°C.
Tissue handling: Removing tile from holder
Seeker 3x3 tile with melted sample is moved to a microcentrifuge tube and submerged in Hybridization Reaction Mix. This step takes place after the CryoCube overlay has been applied (not shown in video). For frozen samples, thaw your tile before this step.
TIPS
- To thaw the tile and holder slide after storing at −80°C, apply a finger underneath the slide to gently heat the tile and sample.
- When gripping the tile, hold the clear, outer section of the tile. Avoid touching the white portion of the tile to prevent the loss of any beads.
Tissue clearing from 3x3 tile: Bead dissociation
Sample tissue and beads are washed from the Seeker 3x3 tile. This step takes place after the tissue clearing reaction has occurred (not shown in video).
TIPS
- It is important to avoid touching the surface of the tile as much as possible.
- It may take a few rounds of washing before you see all the beads fall off the tile. You may see a membrane-like layer remaining on the tile surface after you remove the beads. DO NOT attempt to remove this layer as it will inhibit downstream reactions and prevent you from fully recovering beads in subsequent steps.
Tissue clearing from 10x10 tile: Bead dissociation
Sample tissue and beads are washed from the Seeker 10x10 tile. This step takes place after the tissue clearing reaction has occurred (not shown in video).
TIPS
- It is important to avoid touching the surface of the tile as much as possible.
- It may take a few rounds of washing before you see all the beads fall off the tile. You may see a membrane-like layer remaining on the tile surface after you remove the beads. DO NOT attempt to remove this layer as it will inhibit downstream reactions and prevent you from fully recovering beads in subsequent steps.
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