Figure 2.

HIF-1α and HIF-2α protein detection by peroxidase immunohistochemistry on paraffin-embedded material. COS-1 cells were transfected with either pGN/HIF-1α28–826 (A and E) or pGN/HIF-2α19–870 (B and F) resulting in the expression of fusion proteins in a subset of the cells. Cells were then fixed in formalin and processed into paraffin-embedded blocks in a manner analogous to the handling of diagnostic pathological specimens. Likewise HT1080 cells were cultured in parallel for 4 hours in normoxia or 0.1% hypoxia and processed to produce a normoxic cell pellet (C and G) and a hypoxic cell pellet (D and H). Nuclear staining was observed with mAb 122 (A−D) only in the HIF-1α transfectants (A) and the hypoxic cell pellet (D). mAb 190 detected only the HIF-2α fusion protein (F) and HIF-2α was absent in the normoxic cell pellet (G), but hypoxically inducible (H). The intensity of nuclear staining observed within the hypoxic cell pellet was heterogeneous for both antigens. Original magnifications, ×100 (A, B, E, and F) and ×400 (C, D, G, and H).