Name: detection of neuronal aging in a cortical organoid section
Uploaded: 2025-03-31T15:00:38.000+00:00
Duration: 1 min 15 s
Description: Source: Shaker, M. R., et al. Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro. J. ...

detection of neuronal aging in a cortical organoid section

Detection of Neuronal Aging in a Cortical Organoid Section

After cryosectioning the organoids, remove the excess mounting solution by transferring the slides into a slide standing container with a lid, and wash the sectioned organoid tissue three times with PBS for 10 minutes at room temperature. Then, incubate the slides overnight at 37 degrees Celsius with freshly made beta-galactosidase staining solution. Wash the stained tissues three times with PBS for 10 minutes each at room temperature to remove the beta-galactosidase solution.
Now, mount the washed tissues with a glass antifade mountant and allow the mounting solution to solidify for 30 minutes at room temperature before viewing it under the microscope.

detection-of-neuronal-aging-in-a-cortical-organoid-section

Universidad de Cartagena UDEC

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Neuroscience

Neuropathology

Neurodegenerative Diseases

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Characterization of neuronal aging in Cortical organoids
<ol>
	<li>Process cortical organoids for cryosections: 
	NOTE: The steps were performed in a Class 2 biosafety hood.
	<ol>
		<li>Prepare 2 mL tubes, each filled with 1.5 mL of 4% paraformaldehyde (PFA).</li>
		<li>Cut the end of a P1000 pipette tip (to make it a wide-bore) and gently transfer each organoid to one of the 2 mL tubes prepared above (one organoid per tube). 
		NOTE: To prevent excess Differentiation Medium (DM) media mixing with the PFA, allow the organoid to sink toward the opening of the P1000 pipette tip and rest the tip just above the top of the PFA in the tube before pipetting the organoid out. This will enable the researcher to transfer just the organoid and very minimal media.</li>
		<li>Allow the fixation process to take place at 4&#176;C for 1 h.</li>
		<li>Using an uncut P1000 pipette tip, carefully aspirate excess PFA and add 1.5 mL of cold 1x PBS (Phosphate buffered saline).</li>
		<li>Transfer the tubes to an orbital shaker set at 70 rpm for 10 min at room temperature (RT).</li>
		<li>Repeat the washing process with cold 1x PBS three times to ensure all PFA has been thoroughly removed. 
		NOTE: Do not dispose of PFA in regular waste containers; instead, prepare a specific chemical waste disposal container for this, as PFA is a hazard.</li>
		<li>Immerse the organoids in 1x PBS containing 30% sucrose and incubate at 4 &#176;C until all organoids have sunk to the bottom of the tube. 
		NOTE: The time required for the organoids to sink depends on their size and age. Three-month-old organoids may take up to 5 h.</li>
		<li>Using a cut, wide-bore P1000 pipette tip, gently transfer three to five organoids into a mounting mold containing a mounting solution made of 30% sucrose and 100% optimal cutting temperature (OCT) medium at a ratio of 3:2.</li>
		<li>Use a 10 &#181;L pipette tip with the aid of a stereomicroscope to orientate and position organoids in a grid-like pattern.</li>
		<li>Place the mold on dry ice to solidify the sucrose OCT solution before proceeding with cryo-sectioning (16-20 &#181;m) using a cryostat. 
		NOTE: For senescence-associated beta-galactosidase, all tissues must be processed for sectioning once they have sunk. For immunofluorescence, tissues can be processed for sectioning the next day. If not stained immediately, all the slides containing sections must be stored at -20 &#176;C before subsequent immunofluorescence or beta-galactosidase.</li>
	</ol>
	</li>
	<li>Process for analysis of senescence in the cortical brain organoids: 
	NOTE: The following steps can be performed on a regular lab bench.
	<ol>
		<li>Transfer the slides into a microscope slide staining container with a lid and wash the sectioned organoid tissue three times with 1x PBS for 10 min at RT to remove any excess mounting solution.</li>
		<li>Following this, incubate the washed tissue with freshly made beta-galactosidase staining solution overnight at 37 &#176;C. 
		NOTE: The beta-galactosidase staining solution is made of phosphate buffer (for 10 mL of phosphate buffer: 8.15 mL of 1 M NaH2PO4, 1.85 mL of 1M Na2HPO4) adjusted pH = 6, 100 mM of potassium hexacyanoferrate (III), 100 mM of potassium hexacyanoferrate (II) trihydrate, 5 M of NaCl, 1 M of MgCl2, 20 mg/mL of X-Gal. Avoid using a standard cell culture incubator containing CO2 as the CO2 will alter the pH of the beta-galactosidase staining solution.</li>
		<li>To remove the beta-galactosidase solution, wash the stained tissues with 1x PBS three times for 10 min each at RT.</li>
		<li>Mount the washed tissues with a glass antifade mountant and allow the mounting solution to solidify for 30 min at RT before viewing under the microscope.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16% Formaldehyde (W/V) Methanol-free</td>
			<td>Thermo Fisher Scientific</td>
			<td>28908</td>
			<td>4% of PFA are diluted in 1x PBS</td>
		</tr>
		<tr>
			<td>CKX53 microscope with SC50 camera</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Sigma Aldrich Pty Ltd</td>
			<td>D1408-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21103049</td>
			<td>Used in DM media</td>
		</tr>
		<tr>
			<td>OCT Embedding Compound Sakura Clear (118 mL/Bottle)</td>
			<td>Tissue Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hexacyanoferrate (II) Trihydrate</td>
			<td>Sigma Aldrich Pty Ltd</td>
			<td>CP1087</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hexacyanoferrate(III)</td>
			<td>Sigma Aldrich Pty Ltd</td>
			<td>455946</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Glass Antifade Mountant</td>
			<td>Life Technologies Australia (TFS)</td>
			<td>P36980</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich Pty Ltd</td>
			<td>PHR1001-1G</td>
			<td>30% of sucrose are diluted in 1x PBS</td>
		</tr>
	</tbody>
</table>

Source: Shaker, M. R., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/63714">Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro.</a> J. Vis. Exp. (2022).
This video demonstrates the method of detecting aging neurons in cortical organoid sections by staining for beta-galactosidase activity, which is elevated in senescent cells. The enzyme cleaves a substrate to produce a blue product, followed by microscopic observation of the stained neurons.

Source: Shaker, M. R., et al. Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro. J. ...

Begin with a slide containing a thin section of a cortical organoid that contains glial cells and neurons at various stages of aging.
The aging neurons, in a state of senescence with declining cell function, contain high levels of beta-galactosidase.
The section is pretreated with a fixative to stabilize the cell structure and a permeabilizing agent to permeabilize their membranes.
Place the slide in a staining container to wash with a buffer to remove any solution traces.
Stain the slide with a solution containing a substrate for the beta-galactosidase enzyme and incubate.
The substrate enters the cells, where beta-galactosidase cleaves the substrate, producing a blue-color product that accumulates more in the aging neurons.
Repeatedly wash the section.
Add an antifade mountant and allow it to solidify, ensuring the integrity of the stained section.
Observe the slide under a microscope; the area with an intense blue-color product indicates the aging neurons within the organoid.

8 vues. Detection of Neuronal Aging in a Cortical Organoid Section

Vidéo: Detection of Neuronal Aging in a Cortical Organoid Section - Expérience

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Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

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Detection of Neuronal Aging in a Cortical Organoid Section

Transcription

Detection of Neuronal Aging in a Cortical Organoid Section