When preparing your reference samples, note the following:

  • Define a dye set of n different dyes that are supposed to be used together in a multiplex scan.
  • These dyes should be optimized for the fluorescence channels, ensuring minimal possible crosstalk already in the raw data before unmixing.
  • For each dye of the dye set, one reference sample is required which is a tissue section of a typically used tissue type, stained by only this specific dye (single stained references, no additional nuclear marker).
  • At least one, unstained, reference sample is required of the same tissue type as the dye reference samples. The unstained sample (no nuclear marker) should be processed as similarly as possible like the single-stained references.
  • As an example, a set of six dyes (five specific dyes plus a nuclear marker), the following seven reference samples are required:
    • One sample only stained with dye 1.
    • One sample only stained with dye 2.
    • One sample only stained with dye 3.
    • One sample only stained with dye 4.
    • One sample only stained with dye 5.
    • One sample only stained with the nuclear marker.
    • One sample of the unstained tissue.
  • It is possible to use the same marker for the single-stained reference slides as for the multiplex samples that will be unmixed later. It is favorable, however, to use the same marker for all dyes to ensure a clear and comparable signal at the same sites of the tissue. It also makes the calibration setup easier.