Figure 2.

Fibroblasts obtained from GM1-null mice were cultured with or without 0.3, 3, or 30 μM GM1 for 60 min. (A) Dot blot labeling by b-CtxB. (B) Fluorescence microscopy using b-CtxB, followed by fluorescein isothiocyanate-avidin. The addition of GM1 to the culture medium increases the GM1 content in GM1-null mouse fibroblasts. Scale bar, 10 μm. (C) Freeze-fracture replicas were labeled by anti-GM1 antibody (top row), or by b-CtxB (bottom row). With either probe, labeling was not observed without GM1 loading, and labeling increased according to the amount of loading. Labeling of GM3 was observed without GM1 loading (inset). Ten-nanometer colloidal gold was used for labeling.