FIGURE 7.

Binding of mAbs to PLN sections from various gene-targeted mice. A, frozen sections (7 μm) of PLNs from WT, FucT-IV, and FucT-VII DKO, C1β3GnT and C2GnT-I DKO, GlcNAc6ST-1 and GlcNAc6ST-2 DKO, GlcNAc6ST-1-deficient, and GlcNAc6ST-2-deficient mice were incubated with 5 μg/ml biotinylated S1, S2, or MECA79 for 2 h (red). B, effects of N-glycosidase F treatment on the binding of mAbs or E-PHA to HEVs. Frozen sections from the PLNs of WT and C1β3GnT and C2GnT-I DKO mice were fixed with cold acetone and treated for 24 h at 37 °C with or without 100 milliunits/ml N-glycosidase F (Calbiochem) in 10 mm HEPES-NaOH (pH 7.4), 0.1% Triton X-100, and complete protease inhibitor (Roche Applied Science) and incubated with biotinylated E-PHA (0.1 μg/ml), MECA-79 (1 μg/ml), or S2 (1 μg/ml) for 1 h. The binding of biotinylated E-PHA and mAbs was detected with Alexa Fluor 594-conjugated streptavidin using a confocal laser scanning microscope (LSM510 META). Bar, 50 μm. The data are representative of four (A) or three (B) independent experiments.