Detection of Neuronal Aging in a Cortical Organoid Section

Abstract

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

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Characterization of neuronal aging in Cortical organoids

1. Process cortical organoids for cryosections:
   NOTE: The steps were performed in a Class 2 biosafety hood.
   1. Prepare 2 mL tubes, each filled with 1.5 mL of 4% paraformaldehyde (PFA).
   2. 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 Differentia....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
16% Formaldehyde (W/V) Methanol-free	Thermo Fisher Scientific	28908	4% of PFA are diluted in 1x PBS
CKX53 microscope with SC50 camera	Olympus
Dulbecco's Phosphate Buffered Saline	Sigma Aldrich Pty Ltd	D1408-500ML
Neurobasal Medium	Thermo Fisher Scientific	21103049	Used in DM media
OCT Embedding Compound Sakura Clear (118 mL/Bottle)	Tissue Tek	4583
Potassium Hexacyanoferrate (II) Trihydrate	Sigma Aldrich Pty Ltd	CP1087
Potassium hexacyanoferrate(III)	Sigma Aldrich Pty Ltd	455946
Prolong Glass Antifade Mountant	Life Technologies Australia (TFS)	P36980
Sucrose	Sigma Aldrich Pty Ltd	PHR1001-1G	30% of sucrose are diluted in 1x PBS