Opal four color ihc kit

Paraffin blocks were sectioned and subjected to immunohistochemical staining using the Envision reagent (Dako, K4002). Antigen retrieval was performed by placing sections in Target retrieval solution (Dako, PH6 S1699, PH9 S2367) and heating to 95 °C in a water bath, according to the manufacturer's instructions. For the immunostaining of human gastric cancer specimens, anti‐ST3G5 antibody (Proteintech) was used at dilutions of 1 : 400. In co‐immunostaining experiments, sections were sequentially stained with each antibody using an Opal™ four‐color IHC Kit and fluorescently conjugated tyramide according to the manufacturer's instructions (PerkinElmer, Waltham, MA, USA, NEL794001KT). Horseradish peroxidase (HRP)‐conjugated secondary antibody (GE Healthcare, anti‐mouse IgG: NA931, anti‐rabbit IgG: NA934) was added for 10 min, and incubated with Opal kit working solution including the desired fluorophore. Tissues underwent the microwave treatment for removal of primary and secondary antibodies before another round of staining according to the Opal Multiplex IHC Assay Development Guide and Image Acquisition Information (Akoya Biosciences, Menlo Park, CA, USA).

Paraffin blocks were sectioned and subjected to immunohistochemical staining using the Envision reagent (Dako). Antigen retrieval was performed by placing sections in Target retrieval solution (Dako) and heating to 95 °C in a water bath, according to the manufacturer's instructions. In co‐immunostaining experiments, sections were sequentially stained with each antibody using an Opal™ four‐color IHC Kit and fluorescently conjugated tyramide according to the manufacturer's instructions (PerkinElmer, Waltham, MA, USA). All primary antibodies were used at dilutions of 1 : 500. Horseradish peroxidase‐conjugated secondary antibody (GE Healthcare, Chicago, IL, USA) was added for 10 min and incubated with Opal kit working solution including the desired fluorophore. Tissues underwent the microwave treatment for removal of primary and secondary antibodies before another round of staining according to the Opal Multiplex IHC Assay Development Guide and Image Acquisition Information (Akoya Biosciences, Tokyo, Japan).