The PhenoCycler Fusion protocol used by the Spatial Technologies Unit at the BIDMC for the profiling of lymphatic vessels in the BIDMC - Lymphatic Vessels TMC.
The Phenocycler Fusion (PCF, AKOYA Biosciences) consists of a liquid handling and fluidics unit connected to an integrated brightfield and fluorescent microscope with 5 channels.
Antibodies against proteins of interest are conjugated to a unique barcode, which matches via Watson-Crick base pairing to a respective sequence attached to a fluorescent reporter.
The tissue of interest is incubated with a panel of conjugated antibodies and subsequently exposed to cycles of 3 fluorophores with attached reporters plus DAPI at a time, with stripping of the reporters taking place between cycles. This allows for the imaging of three reporters per cycle.
The first and last cycles are blank and are used to examine the difference in autofluorescence occurring as part of the treatment. DAPI is imaged at every cycle and is used to set the focal plane. Imaging takes place at a constant x20 magnification with brightfield mask taken in advance to highlight the position of the section.