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Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy
Published on: November 8, 2016
Visualization of Ganglioside-Bacteria Interaction Using a Frozen Tissue Overlay Assay with GFP-Labeled Bacteria and
Shinichi Chisada1,2, Kohei Shimizu3,4, Naoyuki Matsunaga5,4
1Kyorin University School of Medicine, Mitaka-shi, Tokyo, Japan. chisada@ks.kyorin-u.ac.jp.
None:
This chapter describes a tissue overlay assay for visualizing ganglioside-bacteria interaction on epithelial cells. Bacteria are labeled with green fluorescent protein (GFP) and incubated with frozen tissue sections pretreated with skim milk to minimize nonspecific binding. Staining actin with Alexa Fluor 568-conjugated phalloidin visualizes the epithelial cell surface. Fluorescence microscopy allows observation of bacterial binding to epithelial cells. To determine whether gangliosides mediate bacterial attachment, ganglioside-specific antibodies are used to block bacterial binding. Additionally, sialidase treatment, which removes sialic acid residues from gangliosides, confirms the specificity of the ganglioside-bacteria interaction.
