Phenoimager ht system

Multiplex immunofluorescence staining was performed with Opal 6-Plex Detection Kit (NEL871001KT; Akoya Biosciences, Marlborough, MA) using 6 Opal dyes along with Spectral DAPI counterstain. FFPE tissue samples were prepared for staining by baking at 60°C for 1 h followed by three 10-min washes in xylene to remove paraffin. Samples were rehydrated via an ethanol gradient into deionized water. A six-step cycle of heat-induced epitope retrieval, blocking, primary antibody incubation, Polymer HRP mouse + rabbit secondary antibody incubation, Opal dye-conjugated tyramide deposition, and antibody stripping was performed for each of the six tissue biomarkers using a Leica BOND RX fully automated research stainer (Leica Biosystems). Slides were mounted with coverslips using ProlongGold and multispectral detection and imaging was performed using the PhenoImager HT System (Akoya). Analysis of the data was performed using InForm cell analysis software. A core biopsy wound is defined as a space or tract surrounded by fibrotic and/or cystic changes with inflammatory changes that follows the shape of the biopsy trocar used in the initial diagnostic biopsy. Adjacent and distant were defined as areas within 400 μm and >3 mm away from the border of biopsy wound, respectively.