Masson s trichrome staining

Assessment of breast cancer biomarkers, including estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor type 2 (HER2), and Ki67 labeling index, was prospectively performed for each patient to guide treatment strategy.^{21 (link)} Immunohistochemical staining was done as previously described.^{22 (link)} Briefly, ER and PgR status were considered positive when there was ≥1% of nuclear staining. HER2 positivity was indicated by 3+ immunohistochemical staining or fluorescence in situ hybridization with a threshold ratio of >2.2.^{23 (link)} Ki67 labeling index was determined by counting at least 500 tumor cells in hot spots. Masson's trichrome staining was performed according to the manufacture's recommended protocol (Sigma–Aldrich, St Lois, MO).
Tumor size was generally assessed based on the pathologically invasive area in the surgical specimen; in patients who received any neoadjuvant therapy, tumor size was assessed by pretreatment magnetic resonance imaging or sonography. Evaluation of axillary lymph node metastasis was pathologically performed and classified according to pTNM staging system (UICC, Seventh Edition, 2009). The pathologists in Kumamoto University Hospital assessed Nuclear Grade based on Atypia score and Mitotic index.

Heart tissues were fixed in 10% neutral buffered formalin for 72 h at room temperature. Histopathological analysis was performed on paraffin-embedded sections of the heart (5 µm) stained with hematoxylin and eosin staining. Briefly, after dewaxing and hydration of paraffin sections, hematoxylin staining was performed for 3 min at room temperature, the sections rinsed with distilled water, and then stained with eosin for 2 min at room temperature and rinsed with distilled water again. Finally, the sections were dried and sealed. Myocardial fibrosis was determined using Masson's trichrome staining (Sigma-Aldrich; Merck KGaA). Briefly, hematoxylin staining for 5 min, ponceau trichrome staining for 10 min and aniline blue staining for 2 min at room temperature. Finally, the sections were dried and sealed. Multiple images were acquired from each stained heart section under a light microscope. The fibrotic areas of paraffin-embedded heart sections were evaluated using ImageJ 6.0 software (National Institutes of Health).