Ab125286

AEP and integrins were detected trough incubation of tissue sections overnight at 4 °C with anti-legumain (Abcam, ab125286), anti-integrin α5 (Abcam, ab150361) and antiintegrin β1 (CST, 9699) antibodies. Next, the sections were incubated with HRP-conjugated anti-goat IgG (R&D Systems, HAF017) and anti-rabbit IgG (CST, 7074) for 60 min at 37 °C. After rinsing three times for 5 min in PBS, the sections were incubated with DAB for 30 s, counterstained with hematoxylin and dehydrated. Evaluation was performed on five fields at ×200 magnification for AEP and integrin expression by two independent researchers. The AEP staining percentage (PP, percentage of positive cells) was scored as 1, 1%-25%, 2, 26%-50%, 3, 51%-75% or 4, 76%-100%. The AEP staining intensity (SI) was scored as 0 (negative), 1 (weak), 2 (moderate) or 3 (strong). Immunoreactive scores (IRS) were obtained by combining the PP and SI scores. The IRS were divided into the following groups: 0-3 (negative), 4-6 (weak positive), 7-9 (positive) and 10-12 (strong positive) [31] .

Tissue microarrays were prepared from both cohorts. The TMA was constructed using a TMA GRAND MASTER 2.4-UG-EN MACHINE, using 1 mm punch sets. Cases with heterogeneous DCIS morphological patterns or grade were sampled from all representative areas. In addition, whole tissue sections from 20 cases compromising 10 pure DCIS and 10 DCIS-mixed cases were assessed to evaluate the pattern of legumain expression in malignant breast tissue and adjacent stroma and normal tissue.
Primary antibody specificity for rabbit polyclonal legumain antibody (ab125286, Abcam, UK) was validated using Western blot on whole-cell lysates of MCF7 and SKBR3 human breast cancer cell lines (obtained from the American Type Culture Collection, Rockville, MD, USA) as previously described [27] [28] [29] . Legumain antibody was used at a dilution of 1:500, which showed a single specific band at the predicted size of 56-kDa.
Expression of legumain protein in DCIS was assessed by immunohistochemistry using the Novocastra Novolink TM Polymer Detection Systems Kit (Code: RE7280-K; Leica, Biosystems, UK). Tissue microarray and full-face sections (4 µm) were stained with rabbit polyclonal legumain (dilution 1:150), and then incubated for 24 h. Normal kidney tissue was used as a positive control, while a negative control was carried out by omitting the primary antibody.