Dab chromogen solution dab substrate kit

Lung specimens were obtained from two patients with classic DM-related ILD (on surgical lung biopsy and autopsy, respectively) and one patient with early-stage lung cancer as a normal control (on lung resection). These specimens had been fixed in 10% formalin and embedded in paraffin. Deparaffinized sections (5 μm thick) were preheated to 120 °C for 20 minutes. After blocking endogenous peroxidase activity with 0.3% H₂O₂ for 20 minutes, the slides were incubated with the rabbit anti-human CD163 monoclonal antibody (1:200; Leica Biosystems, Germany) or isotype control antibody (Jackson ImmunoResearch Laboratories, USA) for 1 h at 20 °C. Subsequently, the sections were incubated with biotin-conjugated gout anti-rabbit IgG antibody (Nichirei, Japan) for 15 minutes, and the immunoreaction was visualized using a 3,3-diaminobenzidine (DAB) chromogen solution (DAB substrate kit; Vector Laboratories, USA), and then counterstained with hematoxylin. Additionally, hematoxylin–eosin staining was also done on another slide from each patient.