Immunohistochemistry

MDA-MB-231 tumors and kidneys were harvested and placed in a 10% formaldehyde solution. Three days prior to sectioning, each organ or tumor was transferred to a 30% sucrose (w/v). All samples were sectioned at 20 µm and subjected to immunohistochemistry using either gamma H2AX (p Ser139) antibody (1:500) raised in rabbit (Novus Biologicals, Cat. No. NB100-384, Littleton, CO, USA), cleaved Caspase 3 antibody (1:1000) raised in rabbit (Novus Biologicals, Cat. No. NB100–56113, Littleton, CO, USA), anti-PCNA [pc10] antibody (1:400) raised in mouse (Abcam, Cat. No. ab29, Cambridge, MA, USA), or anti-vascular endothelial growth factor (VEGF; 1:1000, Abcam Canada, Cat. No. ab1316, Toronto, ON, Canada). Prior to overnight incubation with the primary antibodies at 4 °C, each section was incubated in 0.4% H₂O₂ for 2 minutes, followed by DAKO universal blocking solution (purchased from Diagnostics Canada Inc., Mississauga) for 30 minutes and in normal goat serum for primary antibodies raised in rabbit or normal horse serum for primary antibodies raised in mouse for 30 minutes at room temperature (as per instructions on anti-rabbit Vecstatin ABC Kit [Cat. No. PK-6101] or anti-mouse Vecstatin ABC Kit [Cat. No. PK-6102], Vector Laboratories). The sections were washed in Tris buffered saline (TBS) for 5 minutes. Following the overnight incubation, the sections were washed twice and incubated in biotinylated anti-rabbit IgG (Vector Laboratories, anti-rabbit Vecstatin ABC Kit [Cat. No. PK-6101]) or biotinylated anti-mouse IgG (Vector Laboratories, anti-rabbit Vecstatin ABC Kit [Cat. No. PK-6102]) for 75 minutes. The sections were then incubated in avidin biotin complex (ABC reagent) for 45 minutes at room temperature. Following two additional TBS washes, the sections were subjected to the peroxidase substrate 3, 3′ diaminobenzidine (DAB) prepared as per the instructions in the kit (Vector Laboratories, DAB Peroxidase (HRP) Substrate Kit [Cat. No. SK-4100]). Sections were then counterstained with Hematoxylin Solution, Gill No. 1 (Sigma-Aldrich Canada, Cat. No. GHS116, Mississauga, ON, Canada) and washed twice in tap water and once in 1xTBS for 5 minutes. The sections were then dehydrated in anhydrous ethanol and xylene and were cover-slipped using Permount for visualization under a microscope.