quantification of filamentous actin puncta in rat cortical neurons

Quantification of Filamentous Actin Puncta in Rat Cortical Neurons

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.

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1. Fluorescent Labeling and Immunocytochemistry
Note: The immunofluorescent labeling of primary cortical cell cultures was carried out in glass bottom 35 mm cell culture dishes with a working volume of 1 ml.
<ol>
	<li>Wash the glass-bottom dish twice with phosphate-buffered saline pH 7.4 (PBS).</li>
	<li>Fix cells with 4% paraformaldehyde for 15 min at room temperature (RT).</li>
	<li>Wash cells twice with PBS and permeabilize with 0.1% Triton X-100 in PBS for 5 min.</li>
	<li>Treat cells for 20 min at RT with an F(filamentous)-actin-specific stain, AlexaFluor 488 Phalloidin (1:40, 25 &#181;l of Phalloidin in 1 ml PBS).</li>
	<li>Rinse cells twice with PBS and block with 10% normal goat serum at RT for 1-2 hr.</li>
	<li>Dilute the chicken polyclonal anti-MAP2 (microtubule-associated protein 2) antibody (1:2,500) in PBS with 2% normal goat serum and incubate O/N (overnight) at 4 0C.</li>
	<li>Following incubation, rinse in PBS twice.</li>
	<li>Dilute the secondary antibody (Alexa Red 594-conjugated goat anti-chicken IgG (1:500) with 2% normal goat serum and incubate for 2 hr at RT.</li>
	<li>Rinse with PBS and add 10 &#181;l/dish of Hoescht dye.</li>
	<li>Incubate for 3 min at RT and wash with PBS twice. 
	Note: The labeled cells are now ready for step 2 (F-actin puncta counting).</li>
	<li>Preserve the cell sample with 100 &#181;l of antifade reagent as a coverslipping agent and keep it at 4 0C in the dark for long-term storage. 
	Note: Phalloidin is relatively stable in PBS at +4 &#176;C in the dark for up to 1 week, and fluorescence can be preserved with antifade coverslipping reagents. However, optimal images are obtained from 1-3 days after staining since Phalloidin may start to diffuse out with extended storage.</li>
</ol>
2. F-actin Puncta Counting
<ol>
	<li>Turn on the fluorescent microscope and switch to 20&#215; objective. Open microscope software and set up program at 1280&#215;960 pixel image size and 0.17 &#181;m/pixel image resolution at 1&#215; zoom.</li>
	<li>Acquire images of co-labeled F-actin/MAP2 neurons under green (495 nm) or red (613 nm) fluorescent channels.</li>
	<li>Choose 5 Green (F-actin)/Red (MAP2) immunolabeled/Blue (Hoescht) fluorescent images of individual neurons with clearly defined dendritic arbors.</li>
	<li>Identify the F-actin-rich structures in the second-order dendritic segment (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence.</li>
	<li>Rotate the selected region of images to a horizontal level. Copy and paste as a new image.</li>
	<li>Subtract the background of images, using a consistent adjustment for each dish.</li>
	<li>Count the bright green F-actin puncta and trace the length of the selected dendritic segment manually by trained independent observers. Export the data to a spreadsheet file.</li>
	<li>Calculate the density by dividing the total F-actin labeled puncta (N) by the length (L) of the MAP2 labeled dendrites. Express data as the number of F-actin puncta per 10 &#181;m of dendrite 
	Note: Puncta (size &#8804;1.5 &#181;m) of F-actin fluorescence with a peak intensity of at least 50% above the average intensity of staining in the dendritic shaft were included in each selected dendritic segment.</li>
</ol>

Source: Li, H., et al. <a href="https://app.jove.com/v/53697">Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons.</a> J. Vis. Exp. (2016)
This video demonstrates the procedure for fluorescently staining rat cortical neurons for microscopy. It targets F-actin and dendritic proteins to analyze structural integrity and calculate F-actin density, which is crucial for studying neuronal function and health.

1. Fluorescent Labeling and Immunocytochemistry
Note: The immunofluorescent labeling of primary cortical cell cultures was carried out in glass bottom 35 ...

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&#39;s length to calculate the F-actin density.

9 Views. Quantification of Filamentous Actin Puncta in Rat Cortical Neurons

Quantification of Filamentous Actin Puncta in Rat Cortical Neurons

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