Opal 7 color automation ihc kit

FFPE tissue samples from biopsies of patients enrolled in the MEM-288 clinical trial were immunostained using the PerkinElmer OPAL ^™ 7-Color Automation IHC kit (Cat No. NEL821001KT) on the BOND RX autostainer (Leica Biosystems, Vista, CA). The OPAL 7-color kit uses tyramide signal amplification (TSA)-conjugated to individual fluorophores to detect various targets within the multiplex assay. Sections were baked at 65⁰C for one hour then transferred to the BOND RX (Leica Biosystems). All subsequent steps (e.g., deparaffinization, antigen retrieval) were performed using an automated OPAL IHC procedure (PerkinElmer). OPAL staining of each antigen occurred as follows: slides were blocked with PerkinElmer blocking buffer for 10 min then incubated with primary antibody at optimized concentrations followed by OPAL HRP polymer and one of the OPAL fluorophores. Primary antibodies used for mIF are listed in supplementary Table S1. Individual antibody complexes are stripped after each round of antigen detection. After the final stripping step, DAPI counterstain is applied to the multiplexed slide and is removed from BOND RX for coverslipping. Autofluorescence slides (negative control) were included, which use primary and secondary antibodies omitting the OPAL fluors and DAPI. All slides were imaged with the Vectra^®3 Automated Quantitative Pathology Imaging System.