Fluorescence Immunostaining of Cerebellar Organoid Slices

Take fixed cerebellar organoid sections comprising distinct types of cerebellar neurons.

Wash the sections in buffer to remove residual embedding media.

Incubate in glycine to block residual reactive sites from fixation, reducing non-specific binding during immunostaining.

Add a non-ionic detergent to permeabilize cells.

Wash with buffer, removing the excess detergent.

Now, dry the slide. Transfer it to an immunostaining dish with buffer-soaked paper to prevent tissue drying.

Apply a blocking solution that binds to non-specific sites on the neurons.

Remove the blocking solution. Incubate with primary antibodies, which bind to target antigens within cerebellar neurons.

Wash with buffer, removing unbound antibodies.

Incubate with fluorophore-bound secondary antibodies to bind antigen-bound primary antibodies.

Then, wash with buffer, removing unbound antibodies.

Add a dye to stain the cellular nuclei.

Using fluorescence microscopy, visualize markers expressed in cerebellar neurons, indicating organoid maturation.

Wash the microscope slides containing the organoid sections with 50 milliliters of 1X PBS for five minutes. Then, transfer the slides to a Coplin jar containing fresh 1X PBS.