Figure 4.

Immunolocalization of haptoglobin and its fucosylation state in ovarian carcinoma. A). Detection of total (Hp_T) and fucosylated Hp (Hp_F) in a Hp commercial standard (ST) and a tissue extract from an ovarian tumor developed in Nu/Nu mice (T) by Western blot and lectin binding assays (as described in figure 3). B, C, E). Immunolocalization of Hp, fucosylated Hp and fucosylation. Tissue nuclei were stained with DAPI (blue) to help define the tissues’ structures; Hp was stained with a primary anti-Hp antibody and was developed with a secondary TRITC coupled antibody (red); fucosylation was detected by biotinylated-Aleuria aurantia lectin followed by FITC coupled streptavidin (green). All reagents were used at a 1:100 dilution and the scale bar = 100 μm. B).Histo-immunofluorescence of stromal and epithelial (a) and germinal (b) areas of cancer-free ovarian tissue slices. The analysis of tile scan shows a tridimensional image generated by 25 fields’ caption (left panels); from the tile scan, an area was selected (yellow square) and optical zooms were done twice (10X and 40X). Epithelium (E), stroma (S), and follicles (F). C). Tumor tissue sections from four different patients. Fucosylation of Hp was analyzed in broad zones (5x5) of tissues as a tile-scan analysis. A representative region (yellow box) of each tissue was selected to detect fucosylated Hp in optical zooms. D). Graph showing a semi-quantitative analysis of nuclei, Hp and fucosylation content, considering an average area. This analysis was performed with the Software Zen 2011 (blue edition, Carl Zeiss) determining the mean fluorescence intensity for each molecule of interest given in arbitrary units. E). Immunolocalization of fucosylated Hp in individual cells in an optical zoom from a selected region of a tumor tissue section; the crop section analyzed (yellow box) clearly shows the cytoplasmic distribution of fucosylated Hp with approximately 80% of co-localization found in each selected cell.