Autostainer plus

IHC staining was performed at RT on an automatic staining workstation (Dako Autostainer Plus, Glostrup, Denmark) using anti-SDCBP (ab133267, Abcam, Cambridge, UK), anti-p21 (Clone 4D10; Leica Biosystems NCL-L-WAF-1, Barcelona, Spain) at a 1:10 dilution, anti-E-cadherin (BD Biosciences, CA, USA) at a 1:4000 dilution, anti-Src (active) Clone 28 (#AHO0051, Thermo Fisher Scientific, Waltham, MA, USA) at a 1:300 dilution, anti-Ki67 (clone MIB-1, Dako, Glostrup, Denmark) for 30 min, and anti-Nanog and anti-Notch antibodies, as described. [21 (link)] Counterstaining with Hematoxylin and eosin was the final step. For the Ki-67, p21, and Notch1 proteins, nuclear staining was evaluated and dichotomized as a negative expression (0–10% stained cells) versus positive expression (>10% stained tumor cells). E-cadherin, Src, Nanog, and Notch1 were scored as described [14 (link),21 (link)]. Validation of SDCBP was performed at the nuclear, membrane, and cytoplasmic levels in each patient according to the immunoreactive score (IRS) by multiplying the quantity and staining intensity scores (Table S1).

The formalin-fixed, paraffin-embedded tissues were cut into 3-μm sections and dried on Flex IHC microscope slides (Dako). The sections were deparaffinized with standard xylene and hydrated through graded alcohols into water. Antigen retrieval was performed using Envision Flex Target Retrieval solution, high pH (Dako). Staining was done at room temperature on an automatic staining workstation (Dako Autostainer Plus) with rabbit polyclonal Anti-TMEM16A antibody (Abcam #ab53212) at 1:500 dilution or with rabbit monoclonal Anti-TMEM16A [SP31] antibody (Abcam #ab64085) at 1:100 dilution using the Dako EnVision Flex + Visualization System (Dako Autostainer). Counterstaining with haematoxylin for 1 minute was the final step.
A semiquantitative scoring system based on staining intensity was applied. Immunostaining was scored blinded to clinical data by two independent observers as negative (0), weak to moderately (1+), and strongly positive (2+) based on staining intensity. Scores ≥ 1 were considered as ANO1 positive expression. Cut-off point was determined by survival.