Rat on mouse hrp polymer kit

Paraffin-embedded murine tissues were cut at 3 um and placed on super-frost slides. After dewaxing and rehydration, antigen unmasking was performed with Decloaking Chamber in DIVA Buffer 1X (DV2005L2J Biocare Medical, Pacheco, CA, USA) (3 min at 125 °C, 5 min at 90 °C) (CD31, CD3, Ly6G); for IBA1 staining, antigen unmasking was not performed. Endogenous peroxidases were blocked with 2% H₂O₂ for 20 min and then rodent block M (for IBA1) or PBS/BSA (bovin serum albumin) 2% (for CD31, CD3, and Ly6G staining) were used to block unspecific binding sites. Sections were incubated with the following antibodies: rabbit anti-mouse IBA-1 (1:250, Wako), goat anti-mouse CD31 (1:1000, R&D), rat anti-mouse CD3 (1:1000, Serotec), and rat anti-mouse Ly6G (1:200, BD Biosciences). All the primary antibodies were incubated for 1 h in a humid chamber at room temperature. As secondary antibody, we used a Rat on Mouse HRP polymer kit (Biocare Medical) (CD3, Ly6G), Goat on Rodent (Biocare Medical) (CD31), and Mach1 (Biocare Medical) (IBA1). Reactions were developed with 3,3′-diaminobenzidine, DAB (Biocare Medical) and then counterstained with hematoxylin and mounted with Eukitt.

The slides with tumour tissues were immersed in peroxidase I blocking reagent (Biocare, Medical, USA) for 5 minutes and immersed in preheated Diva Decloaker (Biocare Medical, USA). They were heated in a microwave for 20 minutes. The tissues were then treated with rat anti-CD31 IgG2a (Dianova, Germany) at 4 °C overnight. After washing with PBS for 1 minute for 3 times, a rat-on-mouse-HRP-polymer kit (Biocare Medical, USA) containing rat-on-mouse-HRP-polymer and rat probe was applied according to the procedures recommended by the manufacturer. After washing the slides with PBS for 1 minute for 3 times, pre-warmed 3,3′-Diaminobenzidine (DAB) (Open Biosystems, USA) were added onto the tissues for 5 minutes at 55 °C, and the slides were then immersed with haematoxylin (Thermo Scientific, USA) for 30 seconds. After washing with tap water, they were immersed with 70% and 80% ethanol quickly with shaking. They were then dehydrated gradually in 90%, 100% ethanol twice for 2 minutes, followed by immersed with 100% xylene for 2 minutes for 3 times. The expression of CD31 was visualized on the sections, appeared as brown in colour. Several sections at 2 levels (500 μm between each level) of each tumour tissue were photographed (x100), and the number of CD31 expressed cells was counted per sections in a blinded-manner.