Non specific rabbit igg

Sections (6 μm) were dewaxed with xylene (2 × 4 min), 100% (v/v) ethanol (4 × 1 min), 70% (v/v) ethanol (1 min) and dH₂O (1 min). Antigen retrieval was performed in 0.01 M sodium citrate buffer, pH 6.0 in a steamer for 30 min at 100 °C. Sections were blocked in 3% (v/v) normal goat serum (NGS) (Vector Laboratories, UK) in 0.05% (v/v) PBST for 1 h. Endothelial cells were stained with rabbit anti-CD31 (PECAM) primary antibody diluted 1:100 (Matrigel sections) or 1:200 (4T1.2luc tumours) in blocking buffer overnight at 4 °C. Non-specific rabbit IgG (Dako, Germany) antibody was used as isotype control. Biotin-conjugated goat anti-rabbit secondary antibody (Vector Laboratories, UK) in blocking buffer was incubated for 1 h. Antibody complexes were detected using the ABC reagent kit according to manufacturer’s instructions (Vector Laboratories, UK). Cell nuclei were counterstained with haematoxylin (VWR International, Ireland). Sections were dehydrated in 100% (v/v) ethanol (4 × 1 min) xylene (2 × 4 min) then mounted in DPX mounting medium (Sigma Aldrich, Ireland). CD31 positive endothelial cells were quantified in three fields of view (original magnification 200×) for each Matrigel implant (n = 5/group) or five fields of view (original magnification 400×) for each tumour (n = 3/group) and an average calculated for each group.

Tissue samples were fixed in 10% neutral buffered formalin or 4% paraformaldehyde and embedded in paraffin. Following rehydration and quenching (3% H₂O₂), antigen retrieval was performed by microwaving slides in 10 μM sodium citrate solution for 20 minutes (pH 6). Following the retrieval, slides were blocked using protein blocking solution (DAKO) and then incubated overnight at 4°C with primary antibody (Table 2). For a negative control, non‐specific rabbit IgG (DAKO) was used at the same concentration as primary antibody. Next, sections were incubated with biotinylated secondary antibodies followed by Streptavidin‐HRP. Finally, tissue sections were mounted with Surgipath Micromount^® mounting media (Leica Microsystems Inc.) and examined on an Olympus BX61 Motorized Microscope and MicroSuite^™ system (Olympus America Inc.). The operator was blinded to the treatment groups to minimize operator bias. A minimum of five fields were examined and photographed for each tissue section using Olympus DP72 Microscope Digital Camera (Olympus America Inc.). The number of positively stained immune cells were counted by the blinded operator and recorded to quantify the infiltration of immune cells into the tissue.