Hpa001122

Tissue microarray of TNBC patients with information of clinic-pathological parameters was purchased from Outdo Biotech (HBreD090Bc01; Shanghai, China). Tissue samples were pre-stained with Ki67. All procedures were approved by the Ethical Committee of Tongji Hospital, China. Informed consent was obtained from all subjects. For immunohistochemical staining, antigen retrieval, blocking of non-specific binding and incubation of primary antibodies at 4 °C overnight were conducted sequentially. The primary antibody of anti-WDHD1 (HPA001122, Sigma-Aldrich, 1:500) was used. After incubation with secondary goat anti-rabbit immunoglobulin conjugated to peroxidase-labelled dextran polymer (SV0002; Boster) at 37 °C for 1 h, visualisation, counterstaining with haematoxylin and mounting were performed. Semi-quantitative evaluations of protein expression were scored on the basis of the intensity and the percentage of WDHD1-positive tumour cells as previously described^{59 (link)–62 (link)}.

Cells were fixed in 4% PBS-paraformaldehyde for 15 min, incubated in 0.1% Triton-X-100 for 5 min on ice, then in 0.2% Fish Skin Gelatine in PBS for 1 h and stained for 1 h with an anti-WDHD1 (1:500, Sigma-Aldrich, HPA001122). Protein expression was detected using Alexa Fluor (1:400, Molecular Probes) for 20 min. 4'6-Diamidino-2-Pheylindole (DAPI) (Invitrogen) was used to stain nuclei (1:1000). Samples were observed using a confocal microscope system (Leica SP8). Acquired images were analysed using Fiji^{58 (link)}.