Envision method

Four‐micron sections were cut from formalin‐fixed paraffin‐embedded (FFPE) tissue blocks, heated at 60 °C for 30 min, deparaffinized and rehydrated. These were then subjected to antigen retrieval by heating in epitope retrieval buffer in a 95 °C water bath for 45 min. The slides were incubated with either mouse monoclonal antibodies or rabbit polyclonal antibodies for 30 min at room temperature. Anti‐ColXα1 (1:50, Clone X53, eBioscience/Affymetrix, San Diego, CA, USA) and anti‐alpha elastin (1:200, polyclonal, Abcam, Cambridge, MA, USA) were used for immunohistochemistry (IHC). Immunoreactivity was detected using the DAKO EnVision method according to the manufacturer's recommended protocol. Peri‐ and intra‐tumoral stromal staining for ColXα1 and elastin were scored as 0, 1+, 2+, and 3+ as previous described 17; briefly, 0 for absent staining, 1+ for <5% stroma tissue, 2+ for 5–10% of stroma tissue, and 3+ for >10% of stroma tissue. All scoring was performed blinded to the diagnosis. Developing cartilage and cartilaginous tumors were used as positive controls while normal bone and breast tissue were used as negative controls. Cases with a ColXα1 score 0 and elastin score 1–3+ or vice versa were not considered as coexpression or discordant expression patterns.

Prediluted antibodies for estrogen receptor (ER; clone EP1), progesterone receptor (PR; clone 636) and Ki67 (clone MIB-I) were obtained from Dako (Glostrup, Denmark). Sections were processed in a PT Module using Dako high pH buffer (Dako) for deparaffinization and antigen retrieval. Sections for the Ki-67 study were processed with Dako low pH buffer. All immunohistochemical stains were performed in an Autostainer Link using the EnVision method (Dako). HER2 overexpression was analyzed using the HercepTest assay (Dako). Tumors were classified as ER or PR positive when at least 1% of the tumor cells showed staining in the nuclei cells [29] (link). HER2 was considered overexpressed when a uniform intense (3 +) membrane staining was present in > 30% of invasive tumor cells [30] (link). The percentage of Ki67-stained nuclei was evaluated independently of the intensity and its positivity cutoff value was ≥ 20% [31] (link). Two pathologists independently evaluated all immunostainings, and discordant results were reviewed to reach an agreement.

To investigate human common carotid arteries and the effect of coronary bare metal stents on human coronary arteries by histological and immunohistochemical examinations, 22 samples each that did not show aortitis syndrome and autoimmune disease were obtained at autopsy.
Resected tissues were fixed in 10% formalin and embedded in paraffin. The sections (3 µm) were stained with hematoxylin and eosin (H&E) and used for immunohistochemical examinations by the EnVision method (Dako Japan, Kyoto, Japan). Monoclonal anti-human CD68 (clone KP1, 1:100) and αSMA (clone 1A/4, 1:150) antibodies were obtained from Dako Japan, rabbit polyclonal antibodies to CCL22 (1:100) and monocyte chemoattractant protein (MCP)-1 (1:200) were obtained from Abcam (Tokyo, Japan), and monoclonal anti-human CD4 antibody (clone 4B12, undiluted) was obtained from Nichirei (Tokyo, Japan).
This investigation adhered to all the principles prescribed in the Declaration of Helsinki. The Ethics Committee of Experimentation, University of Occupational and Environmental Health, Japan, approved all procedures of the present study.