Peroxo block solution

Hyaluronan in tumor tissues was localized as previously described [42 (link)], with some modifications. Briefly, five micrometer sections were deparaffinized and rehydrated, and endogenous peroxidase was blocked with Peroxo-Block solution (Invitrogen). Nonspecific staining was blocked using 2% BSA (Jackson ImmunoResearch, West Grove, PA) and 2% normal goat serum (Vector) for 1 h, followed by blocking of endogenous avidin and biotin (Avidin/Biotin Blocking Kit, Invitrogen). Hyaluronan was detected by incubating sections with 2.5 μg/mL bHABP (Seikagaku, Tokyo, Japan) for 1 h at 37°C. Signal was amplified by incubation with streptavidin-horseradish peroxidase solution (HRP; BD Biosciences) and detected with 3,3′-diaminobenzidine (DAB, Dako North America, Carpinteria, CA). Sections were then counterstained in Gill's hematoxylin (Vector) and mounted in Cytoseal 60 medium (American MasterTech, Lodi, CA). Specificity of the staining was assessed by incubation of a section of each sample with rHuPH20 (12,000 U/mL) in PIPES buffer (25 mM PIPES, 70 mM NaCl, 0.1% BSA, pH 5.5) at 37°C for 2 h prior to incubation with bHABP.