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THE FUNDAMENTALS

What is the difference between : number of colors, plex and multiplexing

Number of colors: every single stain on the slide including counterstains

DAPI (counterstain)
DAPI (counterstain)
Cy7
Cy7
Spectrum Green
Spectrum Green
Spectrum Orange
Spectrum Orange

Plex: the number of targets to be analyzed excluding counterstain

2-plex Red and Brown IHC with crystal light green counterstain
2-plex Red and Brown IHC with crystal light green counterstain
2-plex Red and Green RNA ISH with hematoxylin counterstain
2-plex Red and Green RNA ISH with hematoxylin counterstain

Multiplexing: the ability to simultaneously detect 2 or more markers on a single slide (eg CD3, CD4, CD8 & counterstain). The nuclear counterstains most frequently used are hematoxylin for brightfield and DAPI for fluorescent microscopy

Ultivue PD-L1 kit staining human lung, 4-plex plus DAPI
(The protocol was carried out on the BOND RX stainer from Leica Biosystems fully automated stainer and the stained tissue imaged on the Aperio VERSA whole slide scanning system)