3 3 diaminobenzidine substrate kit

Immunostaining was performed to evaluate the localization of CS in the stromal regions of the ECs. Anti-CS mouse monoclonal antibody (CS-56) was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA) and used at a 1:1,000 dilution. To confirm the localization of vasculature in the stromal region, we also performed immunostaining for CD31, a vascular endothelial marker (ready-to-use clone JC70A; Agilent Technologies Denmark ApS, Glostrup, Denmark). First, 4 μm thick sections were cut from the formalin-fixed paraffin-embedded tissues, deparaffinized in xylene, and rehydrated in a graded series of ethanol dilutions. Endogenous peroxidase activity was blocked by incubating the sections in 3% hydrogen peroxide (H₂O₂) for 20 min at room temperature. All sample slides were washed in phosphate-buffered saline (PBS) before being exposed to the primary antibody overnight at 4°C. After washing, the sections were incubated for 30 min at room temperature with Histofine® Simple Stain™ MAX PO MULTI (Nichirei Bioscience Inc., Tokyo, Japan). Positive signals were visualized using a 3,3’-diaminobenzidine substrate kit (Nichirei Bioscience, Inc, Tokyo, Japan.). Tissue sections were counterstained with Mayer’s hematoxylin. A negative control experiment was performed using an antibody diluent that did not contain the primary antibody.