Dako envision flex detection kit

Four micrometre sections were cut from paraffin-embedded blocks of tumours and organs, and mounted on slides. Dewaxed and hydrated sections were stained in Harris haematoxylin solution (Thermo Scientific) and blued with Scott’s tap water substitute (Sigma-Aldrich). Sections were then stained in eosin solution (Sigma-Aldrich), dehydrated and mounted in DPX (VWR International, Lutterworth, UK). For immunohistochemical analyses, freshly cut tissue sections were stained by primary antibodies against carcinoma embryonic antigen (II-7), vimentin (V9) and cytokeratin (AE1-AE3), using Dako Autostainer Link 48 on an automated staining platform and the Dako EnVision FLEX detection kit (Dako, Ely, UK). Slides were counterstained with haematoxylin before dehydration and mounting in DPX (VWR International).

To investigate undiagnosed Lynch syndrome in sporadic bladder cancer, we performed immunostaining for MLH1, MSH2, and MSH6 in an extended population‐based cohort (n = 383) (Sjödahl et al., 2017) using antibodies against MLH1 (ES05, dilution 1 : 50, Dako, Glostrup, Denmark), MSH2 (FE11, dilution 1 : 50, Dako), and MSH6 (EP49, dilution 1 : 50, Dako) and the Dako Envision™ FLEX+ detection kit (Dako) following manufacturer's instruction. In brief, 5 μm sections were deparaffinized and rehydrated and heat‐induced epitope retrieval was performed using a PT Link (Dako) for 20 min at pH 9. The antibody stainings were performed in a Dako Autostainer Plus (Dako) using the Envision™ FLEX+ Mouse or Rabbit LINKER (Dako). Sections were counter‐stained with hematoxylin and eosin, dehydrated, and preserved with coverslips using Permount™ mounting medium (Fisher Scientific, Hampton, NH, USA).