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. 2017 May 19;8(43):73892–73904. doi: 10.18632/oncotarget.18029

Figure 2. Hypoperfusion resulted in tumor necrosis and hypoxia-associated excessive angiogenesis in peri-necrotic region.

Figure 2

The wholly extracted 4T1 tumors were fixed by 4% paraformaldehyde and cut into 6μm thick frozen section for further immunofluorescent staining. 2 h and 15 min before extraction, pimonidazole hydrochloride and TRITC-conjugated lectin was injected into mouse tail vein, respectively. Staining for CD31 (Green), TRITC-conjugated lectin (Red) and pimonidazole hydrochloride (Violet) in (A) tumor edge, (B) non-necrotic and (C) peri-necrotic regions of 4T1 tumors from untreated BALB/c mouse. The necrotic region was surrounded by yellow dotted line. Scale bar: 200 μm. (D) Percentage of perfused-lectin+ CD31+ vessels in all CD31+ vessels of the whole tumor area (Ave indicates average perfusion level), peri-necrotic region (PNR), transitional region (TR) and non-necrotic region (NNR). n = 10. (E and F) Spearman analysis for evaluation of the linear correlation between percentage of hypoxic area (detected by IHC staining for hypoxyprobe) and (E) MVD or (F) vascular branch density in peri-necrotic region (PNR). Quantitative data are indicated as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns indicates no significant (P > 0.05).