Figure 2
From: Diversity in cell surface sialic acid presentations: implications for biology and disease
Some Examples of in situ detection of Sia types and linkages. (a) Differential display of α2-6-linked Sias on the ciliary border of lung bronchial epithelial cells in humans and chimpanzees. Paraffin sections were deparaffinized, rehydrated and blocked for endogenous biotin and overlaid with biotinylated Sambucus Nigra agglutinin (SNA), followed by Tris-buffered saline washes, alkaline phosphatase-labeled streptavidin and additional washes. Color detection used a Vector Labs blue substrate and nuclear fast red counterstain. Arrows point to the epithelial luminal edge, where staining is seen in humans, but not in chimpanzees or in other great apes.18 (b) Expression of 9-O-acetylated Sias in rat liver. Left panel: DFP-treated recombinant soluble influenza C Hemagglutinin-esterase (CHE-FcD). Right panel: non-DFP-treated version of the same probe (CHE-Fc, esterase active, negative binding control).19 Frozen sections of rat liver were blocked for endogenous peroxidases, fixed in formalin, washed, overlaid with the CHE-FcD or Che-Fc probes, which were pre-complexed with HRP anti-human antibodies at predetermined dilutions. After incubation at 4°C for 2 h, sections were washed in PBS and color developed using the AEC substrate with Mayer's hematoxylin for nuclear counterstain. (c) Detection of the nonhuman Sia Neu5Gc in human tumors, using an affinity-purified chicken IgY antibody. Left Panel: example of positive colon carcinoma. Frozen sections of a human colon carcinoma were blocked for endogenous peroxidases, fixed in formalin, washed and overlaid with a chicken anti-Neu5Gc antibody.38 Following washes and incubation with HRP-labeled donkey anti-chicken antibody and further washes, color was developed using the AEC substrate with Mayer's hematoxylin for nuclear counterstain. The right panel shows a negative control using a nonspecific chicken IgY. (d) Detection of GD3 and de-N-acetylated GD3 in human melanomas. Left Panel: detection of GD3 using MAb R24. Right Panel: detection of de-N-acetyl-GD3 using MAb SGR37. Unfixed frozen sections of a human melanoma were blocked for endogenous peroxidases and overlaid with monoclonal antibodies R24 or SGR37.33 Following PBS washes, incubation with HRP-labeled goat anti-mouse Ig antibodies and further washes, color was developed using the AEC substrate with Mayer's hematoxylin for nuclear counterstain. (Note: Scale bar=50 μm for each pair of panels is shown on the left side).
