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. 2020 Jun 30;10:991. doi: 10.3389/fonc.2020.00991

Figure 3.

Figure 3

Evaluation of BM oxygenation using pimonidazole (Pimo) IHC. Mice were euthanized on specified day after injecting the leukemia cells and 3 h after ip injection of pimonidazole (3 mice per group). (A–C) Images of pimonidazole IHC in representative histological sections of formalin fixed paraffin embedded femurs (FFPE, 200x magnification). (A) C57BL6 mouse without leukemia. (B) Intermediate stage of leukemia (day 10+ p.i.). (C) Advanced stage of leukemia (day 14+ p.i.). (D–F) Corresponding IHC intensity and cell/tissue segmentation. Brown +3: high intensity of pimonidazole staining; Orange +2: medium intensity of pimonidazole staining; Yellow +1: low intensity of pimonidazole staining; Blue 0: negative/no intensity of pimonidazole staining. (G) Semi quantitative analysis (Histo-score, H-score) of the intensity of pimonidazole IHC in FFPE femur sections of leukemic and normal mice (Healthy): intermediate (Intermediate) and advanced (Advanced) stages of disease progression (n = 3 mice per time point). (H) H-score analysis of the pimonidazole signal intensity in FFPE skull sections in different stages of disease progression (3 mice per time point). ****p < 0.0001.