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 minutes and label them with an F-actin-specific stain at room temperature. After 20 minutes, rinse the cells two times with PBS and block any nonspecific cell surface staining with 10% normal goat serum.

At the end of the incubation, label the cells overnight at 4 degrees Celsius with chicken polyclonal anti-MAP2 antibody. The next morning, rinse the cells two times with PBS and label them with the appropriate secondary antibody.

After two hours at room temperature, rinse the cells with PBS again and label them with 10 microliters of Hoechst for three minutes at room temperature. Then, wash the cells two final times with PBS and preserve them with 100 microliters of anti-fade reagent.

Within three days of the staining, turn on the fluorescent microscope and select the 20x objective. Next, in the microscope software, set the pixel image size to 1,600 by 1,200 and the 0.17 micron per pixel image resolution to 1x zoom. Transfer the dish of cells onto the microscope stage and acquire images of the co-labeled F-actin MAP2 neurons under the green and red fluorescent channels.

When all of the images have been obtained, choose five images of individual neurons in the green, red, and blue channels with clearly defined dendritic arbors, and use continuous MAP2 immunofluorescence to identify the F-actin-rich structures in the second-order dendritic segments. Rotate the selected region of images horizontally, and copy and paste the region as a new image.

Then, subtract the image background fluorescence and use the software to count the number of bright green F-actin puncta. Use trained independent observers to measure the length of the selected dendritic segments, and then export the puncta numbers and dendritic segment length data to a spreadsheet file.

Be sure that observers include F-actin positive puncta with a peak fluorescence intensity of at least 50% above the average intensity of staining in the dendritic shaft in each selected segment.

Finally, calculate the density according to the formula, expressing the data as the number of F-actin puncta per 10 microns of dendrite.