Envision flex target retrieval solution 3 in 1 ph 9

Four μm TMA-sections were automatically pre-treated with the PT-link system (Agilent Technologies, Santa Clara, CA, USA), including deparaffinization, rehydration and antigen retrieval for 20 min at 97 °C using the EnVision™ FLEX Target Retrieval Solution (3-in-1) pH 9 (Agilent technologies), in 65 °C preheat mode. The slides were then stained in an Autostainer Plus (Agilent Technologies) with a polyclonal antibody (HPA024755, Atlas Antibodies, Bromma, Sweden), dilution 1:200 and incubation time 30 min. The specificity of the antibody to LY6D has been demonstrated in several types of assays in the Human Protein Atlas. The estimated percentage of LY6D positive tumour cells was manually evaluated in each TMA core by three independent observers (NA, JO and KJ), and categorized as 0 (negative), 1 (≤50% positive cells) or 2 (> 50% LY6D positive cells). The staining intensity was not taken into account. Cases with lost cores or an insufficient amount of tumour cells in the TMA cores, as well as cystectomy specimens with no remaining tumour or carcinoma in situ (CIS) only, were excluded from the analyses. The mean score was denoted for multiple cores.