A Sensitive IHC Method for Monitoring Autophagy-Specific Markers in Human Tumor Xenografts

Figure 3

Comparison of LC3B levels in CAIX-positive (hypoxic) and CAIX-negative (normoxic) tumor areas. (a) H&E images Calu-6 xenograft tumor sections from vehicle-treated nude mice. (b) Standard IHC double labeling of CAIX (red) and pS6 (brown) in Calu-6 xenograft tumor sections from vehicle-treated nude mice. CAIX-positive staining (red stain) delineating hypoxic conditions was predominately observed in viable tumor adjacent to necrotic tumor areas (red stain), while the mTOR pathway marker pS6 showed positivity (brown stain) predominately in the normoxic region of tumor determined by CAIX-negative staining. There was very little overlap between the CAIX and pS6 marker staining. (c) Labeling of CAIX and LC3B (left image) was performed on separate Calu-6 xenograft serial sections to annotate CAIX-positive stained areas as hypoxic (H) and CAIX-negative areas as normoxic (N). CAIX-positive and CAIX-negative regions of the tumor sections were manually annotated on to LC3B stained slides indicated with blue lines. Inner circles within the viable tumor delineate areas with no CAIX staining were labeled as normoxic (N). Viable tumor positive for CAIX staining was labeled as hypoxic (H). (d) LC3B positive pixel image analysis results comparing hypoxic (H) regions with normoxic (N) tumor regions showed a significant difference in LC3B level between the two regions ( value = 0.012).

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