Name: Video: Quantification of Filamentous Actin Puncta in Rat Cortical Neurons - Concept
Uploaded: 2025-01-30T14:42:53.000+00:00
Duration: 1 min 24 s
Description: 47 Views. Source: Li, H., et al. Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons. 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.

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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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm Glass Bottom Dishes No. 1.5 coverglass</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-20-C</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 medium</td>
			<td>Life Technologies</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma</td>
			<td>P9155</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 488 Phalloidin</td>
			<td>Life Technologies</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal horse serum</td>
			<td>Life Technologies</td>
			<td>26050-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken polyclonal anti-MAP2</td>
			<td>abcam</td>
			<td>Ab92434</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Red 594-conjugated goat anti-chicken IgG</td>
			<td>Life Technologies</td>
			<td>A11042</td>
			<td></td>
		</tr>
		<tr>
			<td>NucBlue Live cell stain ReadyProbes Reagent Hoescht 33342</td>
			<td>Life Technologies</td>
			<td>R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements software package</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse TE2000-E inverted fluorescent computer-controlled microscope</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of filamentous actin puncta in rat cortical neurons

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.

Quantification of Filamentous Actin Puncta in Rat Cortical Neurons

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.

quantification-of-filamentous-actin-puncta-in-rat-cortical-neurons

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Neurodegenerative Diseases

47 Views. Source: Li, H., et al. Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons. 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. 

Video: Quantification of Filamentous Actin Puncta in Rat Cortical Neurons - Concept

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

JoVE Journal

Immunology and Infection

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Quantification of Filamentous Actin Puncta in Rat Cortical Neurons

Transcript

Quantification of Filamentous Actin Puncta in Rat Cortical Neurons

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)