Name: A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues (Video) | JoVE Journal
Uploaded: 2021-06-03T14:30:00.000+00:00
Duration: 6 min 54 s
Description: Learn a rapid fluorescence-based assay to quantify F-actin in rodent and human brain tissues using phalloidin labeling. This high-throughput method enables direct measurement of actin polymerization status in synaptic samples.

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5 minutes

This work was supported by the Neurological Foundation of New Zealand (1835-PG), the New Zealand Health Research Council (#16-597) and the Department of Anatomy, University of Otago, New Zealand. We are indebted to the Neurological Tissue Bank of HCB-IDIBAPS BioBank (Spain) for human brain tissues. We thank Jiaxian Zhang for her help in recording and editing of the video.

All experimental procedures were carried out in accordance with the regulations of the University of Otago Committee on Ethics in the Care and Use of Laboratory Animals (Ethics Protocol No. AUP95/18 and AUP80/17) and New Zealand legislature. Human brain tissues were obtained from the Neurological Tissue Bank of Hospital Cl&#237;nic-IDIBAPS BioBank in Barcelona, Spain. All tissue collection protocols were approved by the Ethics Committee of Hospital Cl&#237;nic, Barcelona, and informed consent was obtained from the families.
1. Preparation of buffers and reagents
<ol>
	<li>Prepare the following buffers for the homogenization of brain tissue and the preparation of enriched fraction of synaptic terminals: 
	Homogenization buffer: 5 mM HEPES, pH 7.4 supplemented with 0.32 M sucrose 
	Resuspension buffer: 5 mM Tris, pH 7.4 supplemented with 0.32 M sucrose 
	Washing buffer: 5 mM Tris, pH 8.1 
	1.2 M sucrose 
	1.0 M sucrose 
	0.85 M sucrose</li>
	<li>Add homemade or commercial mix of protease and phosphatase inhibitors. 
	NOTE: We have used EDTA-free version of Complete Protease Inhibitor mix (1 tablet per 10 mL buffer) and Phosphatase inhibitor cocktail IV (1:100; volume: volume).</li>
	<li>Prepare the following buffers and reagents for the fixation, permeabilization and binding of phalloidin: 
	Krebs buffer: 118.5 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 2 mM CaCl2, 0.1 mM KH2PO4, 5 mM NaHCO3, 10 mM glucose, 20 mM HEPES, pH 7.4 
	1 M KCl 
	25% glutaraldehyde (stock solution) 
	Krebs buffer containing 0.1 % Triton X-100 and 1 mg/mL NaBH4 
	400x Alexa Fluor 647 Phalloidin in DMSO 
	Krebs buffer containing 0.32 M sucrose 
	50&#160;&#956;M latrunculin A in DMSO (stock solution) 
	1&#160;M KCl (stock solution) 
	CAUTION: Phalloidin is toxic (LD50 = 2 mg/kg) and must be handled with care. Inhalation of glutaraldehyde is toxic and it should be handled in a fume hood.</li>
	<li>Store the buffers at 4 &#176;C and phalloidin and latrunculin A at -20 &#176;C for long-term storage. 
	&#8203;NOTE: Long-term storage of buffers is not recommended. The protocol can be paused here.</li>
</ol>
2. Brain tissue homogenization
<ol>
	<li>Homogenize the cryopreserved or freshly dissected rat brain tissue in 10 volumes of homogenization buffer in a Potter-Elvehjem glass tube and pestle on ice. 
	NOTE: Optimal homogenization is achieved by 15-20 strokes of the pestle by hand for brain tissues. Successful homogenization can be confirmed by smooth flow of the suspension through the glass tube. The protocol can be paused here.</li>
	<li>Determine the protein concentration in the homogenate using a Bradford assay31. 
	NOTE: Alternate homemade or commercial assays for protein estimation can also be used.</li>
	<li>Dilute homogenate samples in Krebs buffer at a concentration of 2-3 mg protein/mL in a volume of 50 &#181;L. 
	&#8203;NOTE: In some of our experiments, we incubate homogenates with 2 &#956;M latrunculin A or DMSO (controls) at 37 &#176;C for 1 h. For this, homogenates are resuspended in 48 &#181;L of Krebs buffer, and 2 &#181;L of 50 &#956;M latrunculin A or 2 &#181;L of DMSO is added. Further for immunoblotting, a small amount of sample (10&#160;&#956;g) is collected after the incubation.</li>
	<li>Fix the homogenate samples (section 5).</li>
</ol>
3. Preparation of isolated nerve terminals
<ol>
	<li>Preparation of synaptosomes 
	NOTE: Alternative protocols32,33 for synaptosomes can also be used.
	<ol>
		<li>Centrifuge brain homogenate at 1,200 x g for 10 min at 4 &#176;C.</li>
		<li>Discard the pellet, which is the crude nuclear fraction.</li>
		<li>Further centrifuge the supernatant (S1) obtained in step 3.1.1 at 12,000 x g for 15 min at 4 &#176;C.</li>
		<li>Remove the supernatant (S2), which is the soluble cytosolic fraction.</li>
		<li>Resuspend the pellet (P2) obtained in step 3.1.3, which is the crude synaptosomal fraction in resuspension buffer. 
		NOTE: The volume of resuspension buffer depends on the amount of starting tissue and the amount of pellet obtained. For example, when starting with 150-300 mg of brain tissues, the pellet obtained can be resuspended in 200 &#181;L of resuspension buffer.</li>
		<li>Load the resuspended crude synaptosomes onto a discontinuous sucrose gradient made of equal volumes of 0.85-1.0-1.2 M sucrose. 
		NOTE: We typically use 1 mL each of the sucrose solution (for 150-300 mg tissue). This can be changed according for larger tissue amounts. Discontinuous gradients can be made using a 25G syringe pressed against the internal wall of the ultracentrifuge tube and gentle layering of the sucrose layers.</li>
		<li>Centrifuge at 85,000 x g for 2 h at 4 &#176;C. 
		NOTE: Because of the high-speed of centrifugation, an ultracentrifuge capable of creating a vacuum to reduce heating is required.</li>
		<li>Collect the synaptosomal fraction at the interface between 1.0 and 1.2 M sucrose using a 200 &#181;L pipet tip.</li>
		<li>Wash the synaptosomal fraction with ice-cold washing buffer by centrifugation at 18,000 x g for 10 minutes at 4 &#176;C. 
		NOTE: For washing, the synaptosomal fraction obtained at the interface of 1.0 and 1.2 M sucrose is collected in a fresh 1.5 mL tube, and an equal volume of washing buffer is added, ensuring the removal of high sucrose from the medium.</li>
		<li>Wash the synaptosomal pellet again with ice-cold homogenization buffer at 12,000 g for 10 minutes at 4 &#176;C.</li>
		<li>Resuspend the synaptosomes in homogenization buffer on ice. 
		NOTE: The protocol can be briefly paused here.</li>
		<li>Determine the protein concentration using a Bradford assay.</li>
		<li>Resuspend the synaptosomes in Krebs buffer at a concentration of 2-3 mg protein/mL in a volume of 50 &#181;L (47.5 &#181;L if synaptosomes are to be depolarized by KCl; see section 4). 
		NOTE: For resuspension, the synaptosomal fraction is first spun at 12,000 x g for 5 min at 4 &#176;C and the supernatant (buffer) is removed. The synaptosomal pellet is then resuspended in Krebs buffer by gentle pipetting using a 200 &#181;L pipet tip.</li>
		<li>Proceed with depolarization (section 4).</li>
	</ol>
	</li>
	<li>Preparation of synaptoneurosomes 
	NOTE: Alternative protocols34,35 for synaptoneurosomes can also be used.
	<ol>
		<li>Pass the brain homogenate through a pre-wetted net filter of 100 &#956;m pore size in a filter holder using a 1 mL syringe. 
		NOTE: Pre-wetting of all filters is important to avoid loss of sample and should be done using homogenization buffer. For this, homogenization buffer is passed through the filters in the filter holder using a 1 mL syringe until the&#160;buffer flow out.</li>
		<li>Collect the filtrate (F1) in a pre-chilled 1.5 mL tube on ice.</li>
		<li>Repeat the process (steps 3.2.1 and 3.2.2)&#160;for F1 fraction to obtain the second filtrate (F2).</li>
		<li>Pass F2 filtrate through a net filter of 5 &#956;m pore size.</li>
		<li>Collect the filtrate (F3) in a pre-chilled 1.5 mL tube on ice.</li>
		<li>Centrifuge filtrate F3 at 1,500 x g for 10 min at 4 &#176;C.</li>
		<li>Resuspend the pellet (synaptoneurosomes) in Krebs buffer on ice at a concentration of 2-3 mg protein/mL in a volume of 50 &#181;L (47.5 &#181;L if synaptosomes are to be depolarized by KCl; see section 4). 
		NOTE: The protocol can be briefly paused here.</li>
		<li>Estimate the protein concentration using a Bradford assay.</li>
		<li>Proceed with depolarization (section 4).</li>
	</ol>
	</li>
</ol>
4. KCl-mediated depolarization of isolated synaptic terminals
<ol>
	<li>Equilibrate synaptosomes/synaptoneurosomes at 37 &#176;C for 5-10 min.</li>
	<li>Stimulate synaptosomes/synaptoneurosomes by adding KCl to increase extracellular K+ to 50 mM for 30 s at 37 &#176;C and add equal volume of Krebs buffer to the respective unstimulated control set. 
	NOTE: For example, add 2.5 &#181;L of 1 M KCl to synaptosomes resuspended in 47.5 &#181;L of Krebs buffer; and add 2.5 &#181;L of Krebs buffer to the respective unstimulated control synaptosome. For experiments wherein a large number of samples are involved, proceed with no more than 2 samples at a time so that the depolarization time does not exceed 30 s.</li>
	<li>Terminate stimulation by adding glutaraldehyde (section 5).</li>
</ol>
5. Fixation and phalloidin staining of samples
<ol>
	<li>Add glutaraldehyde to homogenate/synaptosomal/synaptoneurosomal samples to a final concentration of 2.5% for 2-3 min at room temperature. 
	NOTE: We added 6 &#181;L of 25% glutaraldehyde solution so that the final concentration of glutaraldehyde in the 50 &#181;L samples (homogenates/synaptosomes/synaptoneurosomes) was ca. 2.5%. Fixation is critical and should be fast and hence immediately after adding glutaraldehyde, the sample should be vigorously vortexed.</li>
	<li>Sediment the samples at 20,000 x g for 5 min.</li>
	<li>Remove the supernatant. 
	NOTE: Discard the supernatant in a fume hood as glutaraldehyde is toxic.</li>
	<li>Permeabilize the pellet by resuspension in 100 &#181;L of Krebs buffer containing 0.1% Triton X-100 and 1 mg/mL NaHB4 for 2-3 min at room temperature.</li>
	<li>Sediment the samples at 20,000 x g for 5 min.</li>
	<li>Remove the permeabilization buffer.</li>
	<li>Wash the pellet with 200 &#181;L of Krebs buffer by centrifugation&#160;at 20,000 x g for 5 min.</li>
	<li>Resuspend and stain the pellet with 1x Alexa Fluor 647 Phalloidin (corresponding to 500 &#956;U) in 100 &#181;L of Krebs buffer for 10 minutes in dark at room temperature. 
	NOTE: Other varieties of fluorescent phalloidin analogs are commercially available and can be replaced for the assay. Concentration of phalloidin and total sample volume of incubation may have to be modified according to the amount and type of tissue/sample being tested and optimal conditions should be standardized accordingly.</li>
	<li>Centrifuge the stained samples at 20,000 x g for 5 min.</li>
	<li>Remove the unbound phalloidin (supernatant).</li>
	<li>Wash the sample with 200 &#181;L of Krebs buffer by centrifugation at 20,000 x g for 5 min.</li>
	<li>Resuspend in 200 &#181;L of Krebs buffer containing 0.32 M sucrose. 
	&#8203;NOTE: The protocol can be briefly paused here.</li>
</ol>
6. Fluorometric analysis and light scattering
<ol>
	<li>Dispense Alexa Fluor 647 Phalloidin-stained samples in a black 96-well plate.</li>
	<li>Measure the fluorescence intensity at an excitation wavelength of 645 nm and an emission wavelength of 670 nm in a plate reader at room temperature.</li>
	<li>Transfer the samples from the black 96-well plate to the transparent 96-well plate using 200 &#181;L pipet tips.</li>
	<li>Measure the light scattering at 540 nm to correct for any losses that might have occurred during the previous steps of fixation, permeabilization and staining. 
	NOTE: Variations in biological material retained in the stained samples might be more prominent for smaller amounts of starting material (see Discussion).</li>
	<li>Include a set of Alexa Fluor 647 Phalloidin in Krebs buffer at different concentrations (0.05x, 0.1x, 0.25x, 0.35x, 0.75x, 0.5x and 1x corresponding to 25, 50, 125, 175, 250, 375 and 500 &#956;U) for each batch of the assay as a standard curve. 
	&#8203;NOTE: This is an optional step and does not affect the results of the assay particularly when F-actin levels are being expressed in a relative manner (Section 7). The protocol can be paused here.</li>
</ol>
7. Data analysis
<ol>
	<li>The amount of F-actin in the samples is directly proportional to the fluorescence intensity of bound phalloidin. Express in absolute terms of units of phalloidin bound calculated from the linear curve of the tagged phalloidin standard. 
	NOTE: As an example, see Figures 2A-B, 3A, 4A-B and Supplementary Figure 2.</li>
	<li>Express F-actin levels as a fraction of the control samples. 
	NOTE: As an example, see Figures 5A-B.</li>
</ol>

The authors have nothing to disclose.

The assay described here, essentially adapted from a previous study30 with modifications, employs a phallotoxin, phalloidin tagged with a fluorescent label. Fluorescent phalloidin analogs are considered to be the gold standard for staining actin filaments in fixed tissues47,48,49. In fact, they are the oldest tools to specifically identify actin filaments50 and still remain the mos...

Cytoskeletal protein actin is involved in multiple cellular functions, including structural support, cellular transport, cell motility and division. Actin occurs in equilibrium in two forms: monomeric globular actin (G-actin) and polymerized filamentous actin (F-actin). Rapid changes in the polymerization status of actin (interconversion between its G- and F- forms) result in rapid filament assembly and disassembly and underlie its regulatory roles in cellular physiology. Actin forms the major component of the neuronal cytoskeletal structure and influences a wide range of neuronal functions1,2. Of note, the actin cytoskeleton forms an integral part of the structural platform of the synaptic terminals. As such, it is a major determinant of synaptic morphogenesis and physiology and plays a fundamental role in control of the size, number and morphology of synapses3,4,5. In particular, dynamic actin polymerization-depolymerization is a key determinant of the synaptic remodelling associated with synaptic plasticity underlying the memory and learning processes. Indeed, both presynaptic (such as neurotransmitter release6,7,8,9,10) and postsynaptic functions (plasticity related dynamic remodeling11,12,13,14) critically rely on dynamic changes in the polymerization status of the actin cytoskeleton.
Under physiological conditions, F-actin levels are dynamically and tightly regulated through a multimodal pathway involving posttranslational modification4,15,16 as well as actin-binding proteins (ABPs)4,17. ABPs can influence actin dynamics at multiple levels (such as initiating or inhibiting polymerization, inducing filament branching, severing of filaments to smaller pieces, promoting depolymerization, and protecting against depolymerization), and are in-turn under a stringent modulatory control sensitive to various extra- and intracellular signals18,19,20. Such regulatory checks at multiple levels dictate a strict regulation of actin dynamics at the synaptic cytoskeleton, fine-tuning pre- and postsynaptic aspects of neuronal physiology both at the basal and activity-induced states.
Given the important roles of actin in neuronal physiology, it is not surprising that several studies have provided evidence for alterations in actin dynamics as critical pathogenic events linked to a wide range of neurological disorders including neurodegeneration, psychological diseases as well as neurodevelopmental ailments3,21,22,23,24,25,26,27. In spite of the wealth of research data pointing to key roles of actin in neuronal physiology and pathophysiology, however, significant gaps still remain in the understanding of actin dynamics, particularly at the synaptic cytoskeleton. More research studies are needed to have a better comprehension of neuronal actin and its alterations under pathological conditions. One major area of focus in this context is the assessment of actin polymerization status. There are Western blotting-based commercial kits (G-Actin/F-Actin in vivo assay biochemical kit; Cytoskeleton SKU BK03728,29) and home-made assays for the assessment of F-actin levels6. However, because these require biochemical isolation of F-actin and G-actin and because their subsequent quantification is based upon immunoblotting protocols, they can be time consuming. We herein report a fluorescence spectroscopy-based assay adapted from a previous study30 with modifications that can be used to evaluate both basal levels of F-actin, as well as dynamic changes in its assembly-disassembly. Notably, we have efficiently modified the original protocol that requires samples suitable for a 1 mL cuvette to the current 96-well plate format. The modified protocol has therefore significantly reduced the tissue/sample amount required for the assay. Further, we provide evidence that the protocol is suitable for not only brain tissue homogenates, but also subcellular fractions such as isolated synaptic terminals (synaptosomes and synaptoneurosomes). Lastly, the assay can be employed for freshly dissected rodent brain tissues and long-term stored post-mortem human brain samples. Of note, while the assay is presented in a neuronal context, it can be suitably extended to other cell-types and physiological processes associated with them.

<table><tbody><tr><td>3.5 mL, open-top thickwall polycarbonate tube</td><td>Beckman Coulter</td><td>349622</td><td>For gradient centrifugation (synaptosome prep)</td></tr><tr><td>Alexa Fluor 647 Phalloidin</td><td>Thermo Fisher Scientific</td><td>A22287</td><td>F-actin specific ligand</td></tr><tr><td>Antibody against &amp;nbsp;b-actin</td><td>Santa Cruz Biotechnology</td><td>Sc-47778</td><td>For evaluation of total actin levels by immunoblotting</td></tr><tr><td>Antibody against GAPDH</td><td>Abcam</td><td>Ab181602</td><td>For evaluation of GAPDH levels by immunoblotting</td></tr><tr><td>Bio-Rad Protein Assay Dye Reagent Concentrate</td><td>Bio-Rad</td><td>5000006</td><td>Bradford based protein estimation</td></tr><tr><td>Calcium chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>C3306</td><td>Krebs buffer component</td></tr><tr><td>cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td><td>Sigma-Aldrich</td><td>4693159001</td><td>For inhibition of endogenous protease activity during sample preparation</td></tr><tr><td>Corning 96-well Clear Flat Bottom Polystyrene</td><td>Corning</td><td>3596</td><td>For light-scattering measurements</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td>Krebs buffer component</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D5879</td><td>Solvent for phalloidin and latrunculin A</td></tr><tr><td>Fluorescent flatbed scanner (Odyssey Infrared Scanner)</td><td>Li-Cor Biosciences</td><td></td><td>For detection of immunoreactive signals on immunoblots</td></tr><tr><td>Glutaraldehyde solution (25% in water) Grade II</td><td>Sigma-Aldrich</td><td>G6257</td><td>Fixative</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td>Buffer ingredient for sample preparation and Krebs buffer component</td></tr><tr><td>Latrunculin A</td><td>Sigma-Aldrich</td><td>L5163</td><td>Depolymerizer of actin filaments</td></tr><tr><td>Magnesium chloride hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>M2670</td><td>Krebs buffer component</td></tr><tr><td>Microplates</td><td></td><td></td><td></td></tr><tr><td>Mitex membrane filter 5 mm</td><td>Millipore</td><td>LSWP01300</td><td>Preparation of synaptoneurosomes</td></tr><tr><td>Nunc F96 MicroWell Black Plate</td><td>Thermo Fisher Scientific</td><td>237105</td><td>For fluorometric measurements</td></tr><tr><td>Nylon net filter 100 mm</td><td>Millipore</td><td>NY1H02500</td><td>Preparation of synaptoneurosomes</td></tr><tr><td>Phosphatase Inhibitor Cocktail IV</td><td>Abcam</td><td>ab201115</td><td>For inhibition of endogenous phosphatase activity during sample preparation</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich</td><td>P9541</td><td>Krebs buffer component and for depolarization of synaptic terminals</td></tr><tr><td>Potassium phosphate monobasic ((KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P9791</td><td>Krebs buffer component</td></tr><tr><td>Sodium borohydride (NaBH&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>71320</td><td>Component of Permeabilization buffer</td></tr><tr><td>Sodium chloride (NaCl)</td><td>LabServ (Thermo Fisher Scientific)</td><td>BSPSL944</td><td>Krebs buffer component</td></tr><tr><td>Sodium hydrogen carbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>LabServ (Thermo Fisher Scientific)</td><td>BSPSL900</td><td>Krebs buffer component</td></tr><tr><td>SpectraMax i3x</td><td>Molecular Devices</td><td></td><td>For fluorometric measurements</td></tr><tr><td>Sucrose</td><td>Fisher Chemical</td><td>S/8600/60</td><td>Buffer ingredient for sample preparation</td></tr><tr><td>Swimnex Filter Holder</td><td>Millipore</td><td>Sx0001300</td><td>Preparation of synaptoneurosomes</td></tr><tr><td>Tissue grinder 5 mL Potter-Elvehjem</td><td>Duran Wheaton Kimble</td><td>358034</td><td>For tissue homogenization</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td>Component of Permeabilization buffer</td></tr><tr><td>Trizma base</td><td>Sigma-Aldrich</td><td>T6066</td><td>Buffer ingredient for sample preparation</td></tr></tbody></table>

a time-efficient fluorescence spectroscopy-based assay for evaluating actin polymerization status in rodent and human brain tissues

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

Linearity of the assay for evaluation of F-actin levels 
First, a standard curve for the linear increase in fluorescence of Alexa Fluor 647 Phalloidin was ascertained and was repeated for each set of experiments (Figure 1). To investigate the linear range of the assay, different amounts of brain homogenates from rodents (Figures 2A and 2B) and post-mortem human subjects (Figure 3A and 3B) wer...

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

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Learn a rapid fluorescence-based assay to quantify F-actin in rodent and human brain tissues using phalloidin labeling. This high-throughput method enables direct measurement of actin polymerization status in synaptic samples.

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues (Video) | JoVE Journal

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

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