Quantification of Filamentous Actin Puncta in Rat Cortical Neurons

Take rat primary cortical neurons in a polymer-coated glass-bottomed dish.

Fix the neurons with paraformaldehyde to preserve cellular structures.

Wash off the excess paraformaldehyde.

Add a non-ionic detergent to permeabilize the cells.

Treat the cells with a green fluorescent stain targeting the structural protein filamentous actin, or F-actin.

Remove the excess stain.

Apply a blocking agent to prevent non-specific antibody binding.

Incubate with primary antibodies that target dendritic proteins.

Remove the unbound antibodies.

Add red fluorophore-conjugated secondary antibodies to bind to the primary antibodies. Wash off the excess antibodies.

Apply a fluorescent dye to stain the nuclei. Remove the unbound dye.

Add an antifade reagent to prevent fluorophore photobleaching.

Using fluorescent microscopy, capture images of the neurons.

Identify an F-actin-rich region within the labeled dendrite.

Using software, count the F-actin puncta or spots and manually trace the dendrite's length to calculate the F-actin density.

Fix the cells with 4% paraformaldehyde for 15 minutes at room temperature, followed by two PBS washes. Next, permeabilize the cells with 0.1% Triton X-100 in PBS for 5