Figure 5

Immunohistochemistry staining pattern of metabolic (CD31, CAIX), hypoxic (HIF-1α, Hypoxyprobe), and tissue morphology markers. Microscopic images of MB49 tumor tissues treated with nanobubble shell (CTRL) or oxygen nanobubbles (Treatment): Expression of HIF-1 (immunohistochemistry, magnification ×40, scale bar = 100 µm) in CTRL (a) and ONB (b) treated tumors shows strong HIF-1 expression in control tumors and weak positive expression in ONB treated tumors. Representative IHC images for control (c) and ONB (d) treated tumor tissues show that CD31 expression levels were significantly lower in ONB treated groups compared to control (P < 0.05). Representative images of hypoxia immunostaining for Hypoxyprobe-1 adducts and tissue necrosis in tumor sections for control (e) and ONB-treated (f). Representative images of CAIX staining in tumor sections for control (g) and ONB-treated (h). H&E-stained histology images of the corresponding tumor sections showing significantly lower tumor cell volume (% cell volume/mm² of tumor mass) for mice treated with ONB (j) compared to sections of tumors injected with ONB shell (i). Images captured at ×40 objective magnification. Scale bar = 100 µm. HIF-1 protein localization analysis by immunohistochemistry showing significant reduction in the number of HIF-1 positive cells per mm² of tumor area with ONB treatment (k). Histomorphometric quantification of CD31 vessel density (l).