Lectin staining.
FLUOR
MOA, WGA, and UEA-I lectins have binding specificities for carbohydrate sequences present in the glycocalyx. MOA lectin binds to nonreducing terminal galactose-α-1,3-galactose-carbohydrates, WGA lectin binds to the sialyloligosaccharides, N-Acetylglucosamine and N-Acetylneuraminic acid, and UEA-I binds to α-L-fucose. Lectins were labeled with biotin or FlTC: biotinylated MOA (Z8-BA-9001-1, TCS Biosciences, 2 mg/mL; 1:100); FITC-WGA (GTX01502; GeneTex; 5 mg/mL; 1:500); and biotinylated UEA-I (GTX01511; GeneTex; 2 mg/mL; 1:200):Paraffin-embedded kidney sections (5 μm) were dewaxed in Histo-Clear II (National Diagnostics) followed by rehydration in graded ethanol and a wash in PBS. All sections were incubated in blocking buffer (1% BSA in PBS containing 0.1% Tween) for 30 minutes. For biotinylated lectins, this was followed by endogenous biotin blocking using a streptavidin/biotin blocking kit (SP-2002; Vector Laboratories). After 2 washes, the sections were incubated with the biotinylated lectin (pH 6.8) overnight at 4°C. Buffer only was used as a negative control. After 4 washes, the sections were incubated with streptavidin–Alexa Fluor 488 (1:500, S32354; Thermo Fisher Scientific) (pH 6.8) for 1 hour at room temperature. For FITC-labeled lectins, the sections were incubated overnight at 4°C (pH 6.8). Then, for all sections, the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (D1306; Thermo Fisher Scientific) and the cell membrane was labeled with R18 (1:1,000, O246; Thermo Fisher Scientific) for 10 minutes at room temperature. After a 2-minute wash in PBS, the coverslips were mounted in Vectashield mounting medium (H-1000; Vector Laboratories) and examined using either an AF600 LX wide-field fluorescence microscope (Leica Microsystems) or a Leica SP5-II confocal laser scanning microscope attached to a Leica DMI 6000 inverted epifluorescence microscope.