ArticleMethods in molecular biology (Clifton, N.J.)2023
Retinal Ganglion Cell Axon Fractionation.
Article in Methods in molecular biology (Clifton, N.J.), 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Retinal ganglion cell (RGC) axon regeneration in mammals can be stimulated through gene knockouts, pharmacological agents, and biophysical stimulation. Here we present a fractionation method to isolate regenerating RGC axons for downstream analysis using immunomagnetic separation of cholera toxin subunit B (CTB)-bound RGC axons. After optic nerve tissue dissection and dissociation, conjugated CTB is used to bind preferentially to regenerated RGC axons. Anti-CTB antibodies crosslinked to magnetic sepharose beads are used to isolate CTB-bound axons from a nonbound fraction of extracellular matrix and neuroglia. We provide a method of verifying fractionation by immunodetection of conjugated CTB and the RGC marker, Tuj1 (β-tubulin III). These fractions can be further analyzed with lipidomic methods, such as LC-MS/MS to gather fraction-specific enrichments.
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