This article describes a method for identifying aging neurons within cortical organoids using beta-galactosidase staining. The process highlights the cellular changes associated with aging and senescence in neuronal cells.
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.
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.
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