This article describes a detailed protocol for the biotinylation and visualization of sialoglycoproteins in primary neural stem and progenitor cells. The method involves fixation, biotinylation, and immunostaining, followed by fluorescence microscopy to observe the labeled proteins.
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.
First, remove the inserts from the coculture plates, and aspirate the culture medium from the bottom of the wells. Next, wash the neural cells once with prewarmed PBS. Aspirate the PBS from the wells, and add 1 milliliter of a prechilled 4% paraformaldehyde in PBS to each well.
Fix the cells at room temperature for 10 minutes. Then, wash the cells three times with prechilled PBS. Aspirate the PBS, and add 1 milliliter of freshly prepared biotin-conjugated buffer I into each well. Incubate at room temperature for 10 minutes. After this, aspirate the reaction buffer from the wells, and wash the cells three times with PBS. Add 1 milliliter of staining buffer to each well of cells, and incubate at room temperature for 30 minutes.
Next, aspirate the staining buffer from the wells, and wash the cells three times with prechilled PBS. Add 1 milliliter of blocking buffer to each well of cells, and incubate at room temperature for 10 minutes. Remove the blocking buffer from the wells, and add 1 milliliter of primary antibody solution to each well. Incubate the cells overnight at 4 degrees Celsius.
The next day, remove the primary antibody solution from the wells. Wash the cells three times with prechilled PBS. Aspirate the PBS from the wells, and add 1 milliliter of secondary antibody to each well. Incubate the cells at room temperature for two hours. After this, aspirate the solution from the wells, and wash the cells three times with prechilled PBS.
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