Opal 7 colour manual ihc kit

To evaluate the localization and abundance of multiple immune cells in PDAC tissues, fluorescent mIHC was performed in serial sections of FFPE tumor tissue from each PDAC patient using the Opal 7-Colour Manual IHC Kit (PerkinElmer, Hopkinton, Massachusetts, USA) according to the manufacturer’s protocol. In each section, 3–5 markers of interest were stained simultaneously using dyes with different fluorescence signals (Opal 520, Opal 570, Opal 620 and Opal 690), and the nuclei were counterstained by DAPI (4′,6-diamidino-2-phenylindole, a blue-emitting fluorescent compound used for nuclear staining). Scanning and analysis of mIHC slides were performed on a Vectra Polaris Automated Quantitative Pathology Imaging System (PerkinElmer, Boston, Massachusetts, USA).

For the detection of low abundance proteins, TSA signal amplification was performed with the Opal 7‐colour manual IHC kit (Perkin Elmer, Waltham, MA, USA). Deparafinisation, blocking of endogenous peroxidase, and antigen retrieval in citrate buffer were performed as described previously. Slides were incubated for 1 h with anti‐PD‐L1 (Clone SP142, Spring Bioscience, Pleasanton, CA, USA) followed by a 30 min incubation with poly‐horseradish peroxidase solution (Immunologic). Fluorescence detection was achieved by using the Opal 520 reagent (Perkin Elmer). Finally, after microwave‐mediated antibody stripping, the slides were sequentially incubated with primary antibodies, secondary fluorescent antibodies, and directly conjugated antibodies (Table 1), using the previously described method.

The abundance of TITreg cells was analyzed utilizing Opal 7-Colour Manual IHC Kit (PerkinElmer NEL811001KT) according to the manufacture’s protocol (14 (link)). In brief, the slides were incubated with Antibody Diluent blocking buffer (PerkinElmer) at room temperature (RT) for 10 min. The primary antibody for CD4 (Abcam, ab133616, 1:500) and FoxP3 (R&D, MAB8214, 1:400) were incubated at RT for 1 h. Then, a secondary HRP antibody were incubated at RT for 10 min. Signal amplification was performed using Opal 520 TSA (PerkinElmer) and incubated at RT for 10 min. Visualization of the slides was done using the Mantra Quantitative Pathology Imaging System (PerkinElmer) and analyzed using InForm Image Analysis software (PerkinElmer, version 2.1). The TITreg cell fraction in CD4⁺ cells were calculated and data were presented as mean ± SD.