Immunofluorescent Staining of Sialoglycoproteins in Neural Progenitor Cells and Neurons

Take primary neural stem and progenitor cells and neurons, which have sialoglycoproteins tagged by a sialic acid reporter. 

Add paraformaldehyde to fix the cells. Remove excess paraformaldehyde using buffer.

Incubate with a reaction mixture containing biotin-based chemical probes, a reducing agent, and a metal-based catalyst.

The reducing agent activates the catalyst, facilitating the biotinylation of sialoglycoproteins.

Remove the reaction mixture and wash with buffer. Add fluorophore-conjugated streptavidin to bind the biotinylated sialoglycoproteins.

Remove the unbound streptavidin and wash with buffer. Add proteins and a non-ionic detergent to block non-specific sites and permeabilize cells.

Remove this solution and incubate with primary antibodies targeting specific cellular proteins.

Remove the unbound antibodies.

Incubate with fluorophore-tagged secondary antibodies and a fluorescent dye to bind protein-bound primary antibodies and label the nuclei.

Remove unbound molecules. Wash with buffer.

Using fluorescence microscopy, image the cells to visualize the fluorescent sialoglycoproteins and cellular proteins.