Discovery auto stainer

All pathological specimens were reviewed by experienced pathologists who determined primary tumor characteristics based on biopsy specimens obtained for BC diagnosis. Pathologists determined BC histology and receptor status (estrogen receptor [ER], progesterone receptor [PgR], and human epidermal growth factor receptor-2 [HER2]) according to hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining (17 (link)). In terms of Ki-67, pathologists assessed it by IHC on the Ventana Discovery autostainer using the antibody MIB-1 as previously described (18 (link)). For semiquantitative analysis, signals for Ki-67 were graded by two expert pathologists as follows: 0–25%, 1+; more than 25–50%, 2+; more than 50–75%, 3+; more than 75%, 4+. Histologic grade and nuclear grade were also evaluated by Bloom–Ricardson grading and the World Health Organization grading system (19 (link)).
Pathologists determined the pathological response to NAC using surgical specimens. Pathologic complete response was defined as no residual invasive tumor in both the primary tumor bed and ipsilateral axillary lymph nodes (ypT0/Tis, N0) (20 (link)). An RCB class was also calculated based on pathological characteristics at surgery (9 (link)).

Immunohistochemistry was performed using a Discovery Auto-Stainer with automated protocols (Ventana Medical Systems, Inc., Tucson, AZ, USA; Roche, Mannheim, Germany; Ventana Discovery XT, NexES version 10.6 software, DAB-Map kit 760 − 124), as previously described [22 (link), 28 (link)]. The pAMPK antibody (rabbit monoclonal, 40H9, 2535, and 1:300) was purchased from Cell Signaling Technology, Inc. (MA, USA). Total AMPK antibody (mouse monoclonal, D-6, sc-74,461, 1:50), PPARg antibody (mouse monoclonal, E-8, sc-7273, 1:50), and PPARa antibody (mouse monoclonal, H-2, sc-398,394, 1:50) were purchased from Santa Cruz Biotechnology Inc (TX, USA). We have previously clarified the specific immunostaining for pAMPK and total AMPK antibodies in the liver and heart [24 (link)]. We performed negative control staining and confirmed that no primary antibodies were detected in tissues without secondary antibodies.

Immunohistochemistry (IHC) was performed using Discovery Auto-Stainer with automated protocols from formalin-fixed paraffin-embedded mouse sections (Ventana Medical Systems, Inc., Tucson, AZ, USA; Roche, Mannheim, Germany). The primary antibodies used were anti-Ki67 antibody (#518–102456) from Roche (Mannheim, Germany) and anti-cleaved caspase 3 antibody (1:200 dilution; #9661) from Cell Signaling Technology.

Ninety-one formalin-fixed, paraffin-embedded archived surgical specimens of human prostate cancer were randomly selected for analysis by IHC. Antibodies were used at the following dilutions: 1:50 for YB-1 (Cell Signaling Technology, Danvers, MA, USA); 1:50 for EGFR (Abcam, Cambridge, MA, USA) and 1:50 for CXCL14 (Cell Signaling Technology, Danvers, MA, USA). A Discovery Auto-Stainer was employed to perform IHC according to the automated protocols (Ventana Medical Systems, Inc., Tucson, AZ, USA). The intensities of YB-1, EGFR and CXCL14 were determined by qualitative assessment of two levels: negative and positive. The intensity of YB-1, EGFR and CXCL14 staining was scored as: 0 (no staining), 1 (weak staining), 2 (moderate staining) or 3 (strong staining). The percentages of staining were scored as: 0: 0%, 1: 1–25%, 2: 26–50%, 3: 51–75%, and 4: 76–100%. A YB-1, EGFR and CXCL14 status (score ≤ 4) was defined as negative while scores > 4 represent positive. All proceedings have been conducted in compliance with the Human Genome/Gene Research Ethical Guidelines and were approved by the Ethics Committee of Sichuan Cancer Hospital (Approval Number SCCHEC-03-2018-005).