Chst15

The following primary Abs were used: CHST15 (GeneTex), F4/80 (Serotec), and αSMA (Dakop) for paraffin-embedded sections and CS-56 (Seikagaku) for frozen sections. Frozen lung sections were prepared as described previously.³⁵ (link) CS-56 recognizes an epitope on some intact chondroitin sulfate glycosaminoglycan chains. Immunolabeling for F4/80 was performed as reported earlier.7 (link), 36 (link) Immunolabeling for CHST15 was performed with anti-human CHST15 Ab followed by EnVision+ System horseradish peroxidase (HRP)-labeled anti-rabbit polymer (Dako) after antigen retrieval, incubation of the lung section with Target Retrieval Solution (pH 9; Dako) for 15 minutes at 121°C, and staining with 3,3′-diaminobenzidine (Dako). Immunolabeling for CS-56 and αSMA was performed using a Histofine kit (Nichirei Bioscience) according to the manufacturer’s instructions. The number of F4/80⁺ cells was determined using light microscopy in at least 20 randomly chosen high-power fields.6 (link), 36 (link) For quantification of immunostained areas (CHST15, CS-56, and αSMA), bright-field images were captured using a digital camera at 100-fold magnification and the positive areas in five fields per section were measured using ImageJ software.14 (link), 37 (link)