Rat on mouse hrp polymer and probe

Tumor sections were preserved in OCT compound, frozen and sectioned. 5 μm thick sections were fixed in 35/65 methanol/acetone. CD31 was detected with anti-mouse CD31 antibody (MEC13.3, Biolegend, San Diego CA). For the remaining IHC staining, Formalin-fixed tissue samples were paraffinized and sectioned by University of Iowa Comparative Pathology Core. Sections were then de-paraffinized in a series of xylene washes and rehydrated with ethanol and water. Antigen retrieval was accomplished by immersing slides in Citrate Buffer pH 6.0 in a decloaker (Biocare Medical, Concord CA). Tissue samples were incubated with 3% hydrogen peroxide followed by blocking with horse serum (Biocare Medical). Slides were then incubated with rabbit anti-mouse Ki-67 antibody (D2H10, Cell Signaling Technology), rat anti-CD34 antibody (MEC14.7, Novus Biologicals, Littleton CO) or rabbit anti-HIF1α (polyclonal, Abcam, Cambridge UK). Slides were then stained with Rat-on-mouse HRP polymer and probe (Biocare,) or secondary rabbit envision (DAKO, Santa Clara CA). The slides were developed with 3,3’-diaminobenzadine (DAB and 0.3% H₂O₂ in PBS) developing buffer, followed by counterstaining with hematoxylin. Positive staining was quantified using Image J software. All analysis of IHC was done blinded in regards to group allocation.