Opal 5 color manual ihc kit

Opal multiplex staining was performed according to the Opal 5-Color Manual IHC Kit (PerkinElmer). Opal DAPI, Opal 520, Opal 570, and Opal 650 from Abcam were used to generate different signals corresponding to Ku70, VAV2, or Ku80 antibody, respectively. The slides were counterstained with DAPI for nuclear visualization and subsequently coverslipped using VectaShield Hardset mounting media. The slides were imaged using a Vectra Polaris Automated Quantitative Pathology Imaging System (PerkinElmer). We used inForm software (PerkinElmer) to unmix and remove autofluorescence and to analyze the multispectral images.

Formalin-fixed paraffin-embedded (FFPE) sections of esophageal precursor lesions were collected from 30 patients between 2016 and 2018 in Linzhou Cancer Hospital, including INF (n = 5), HYP (n = 13), DYS (n = 8) and CIS (n = 3), to validate the results obtained in mice. The protein expression levels of the marker genes were detected by IHC staining for mice tissues and immunofluorescence for human specimens with antibodies (Abcam) shown in Supplementary Table 3. The samples were incubated with antibody against Ki67 (1:50 for IHC, ab16667), Top2a (1:8000 for IHC, 1:10,000 for IF, ab52934), Aldh3a1 (1:200 for IHC, 1:600 for IF, ab76976), Atf3 (1:200 for IHC, 1:600 for IF, ab216569), S100a8 (1:500 for IHC, 1:1500 for IF, ab92331), Mmp14 (1:2000 for IHC, 1:6000 for IF, ab51074), or Itga6 (1:250 for IHC, 1:750 for IF, ab181551). Opal multiplex staining was performed according to the Opal 5-Color Manual IHC Kit (Perkin Elmer). Opal DAPI, Opal 520, Opal 570, Opal 620, and Opal 690 were used to generate different signals. Slides were counterstained with DAPI (1:2000) for nuclei visualization, and subsequently coverslipped using a VectaShield Hardset mounting media. The slides were imaged using Vectra Polaris Automated Quantitative Pathology Imaging System (Perkin Elmer). We used inForm software (Perkin Elmer) to unmix and remove autofluorescence and to analyze the multispectral images.