Publication protocol
Indirect detection by fluorescence was based on the OpalTM Multiplex IHC method (PerkinElmer/Akoya, USA), and performed on the Autostainer Link 48 system (Agilent/Dako, Denmark) with a PT link module to standardize the staining process. Deparaffinization, antigen retrieval, and antibody stripping were carried out for 20 min at 97 °C using the EnVision™ FLEX Target Retrieval Solution (3-in-1) pH 9 (Agilent/Dako), in 65 °C preheat mode. Subsequent staining was performed using the OpalTM 4-Color Manual IHC Kit (PerkinElmer/Akoya, USA) according to the manufacturer’s recommendations. Signal amplification and covalent binding of fluorophore was achieved by using a tyramide signaling amplification reagent (included in the Opal kit) that is conjugated with a different fluorophore for each cycle [8]. Each fluorescent stain performed included markers for epithelial tissue and DAPI (described further below). Thus, in a 3-plex stain there is room for analysis of one biomarker, in a 4-plex there is room for two, and in a 5-plex there is room for three. A total of three 3-plex stains (for analysis of SOX9, β-catenin, and E-cadherin), one 4-plex stain (for analysis of CDX2 and SOX2), and one 5-plex stain (for analysis of CDX2 and two unpublished markers) were performed in the study (see also Table 2 for a list of all stains and Table S1 for an overview of the staining procedure for each multiplex stain and included biomarkers). Tissue samples were incubated for 30 min with the following primary antibodies: CDX2 (1:50, clone 88, Abcam, UK; detected by Opal 520 at 1:100), SOX2 (1:25, clone SP76, Cell Marque/Sigma-Aldrich, Germany; detected by Opal 570 at 1:100), SOX9 (1:500, Sigma-Aldrich; detected by Opal 570 at 1:100), E-cadherin (1:16000, clone 36, Becton Dickinson, USA; detected by Opal 570 at 1:100), and β-catenin (1:3000, clone 14, Becton Dickinson; detected by Opal 570 at 1:100). In the last cycle of antibody staining, the tissue was hybridized with a cocktail of epithelial markers to allow for complete and accurate epithelial segmentation by the DIA algorithm (anti-pan Cytokeratin (1:1500, clone C-11, Abcam) and anti-pan Cytokeratin Type I/II (1:1000, clone AE1/AE3, Thermo Fisher Scientific, USA); these were detected by Opal 670 at 1:100. For the 4- and 5-plex stains, anti-E-cadherin (1:16000, Clone 36, Becton Dickinson) was included in the epithelial antibody cocktail. Counterstaining was performed using DAPI (PerkinElmer/Akoya) according to the manufacturer’s protocol. Finally, the slides were mounted using ProLong Diamond Antifade Mountant (Invitrogen/Thermo Fisher Scientific). A separate single-plex stain was performed for each fluorophore to create spectral libraries for unmixing of individual spectral signatures in the multiplex. In addition, one slide was not probed with any fluorophore, thus providing the spectral signature of the tissue autofluorescence. The chosen concentration of antibodies was based on optimizing the staining specificity, signal intensity, and signal-to-noise level for both chromogenic DAB and fluorescence staining among control tissues embedded on a separate test TMA, including 42 primary colorectal cancer cases and six samples from normal colon mucosa (Fig. S2). Fluorescence signal intensities for all markers were balanced and kept within the recommended signal range for optimal spectral unmixing of fluorophores with the Vectra 3 system, at between 0 to about 30 counts with the UV lamp power set to 10%. In addition, a negative control experiment where the primary antibody was omitted was performed. To confirm that antibodies were properly stripped away or denatured between cycles [15], the following control experiment was performed for each antibody: after deparaffinization/antigen retrieval, sections were probed with the antibody and stained with its paired fluorophore. This was then followed by heat-treatment in the PT-link. A new round of staining was then performed, however, this time omitting any primary antibody and applying a different fluorophore after secondary antibody incubation. The tissue was imaged and analyzed to confirm that there was no signal above noise originating from the second fluorophore applied. Uniformity of staining was assessed visually and by scatterplots and Spearman’s correlation coefficients assessing the association between protein expression and sample age (Fig. S3).
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