A protocol for isolation and biochemical characterization of stigmoid bodies from rat brain.
Stigmoid bodies (SBs) are structures present in the cytoplasm of neurons. Many brain regions including hypothalamus, thalamus, amygdala, septum, hippocampus, colliculi, and brainstem contain neurons with at least one SB. Despite this widespread distribution their function remains unknown. SBs contain a brain protein called huntingtin-associated protein 1 (HAP1) and have more recently been found to contain the apolipoprotein E receptor LR11 (Lipoprotein Receptor containing 11 LDL binding domains, also called SorLA for sorting protein-related receptor containing LDLR class A repeats) and sortilin. To provide a first step towards further identification of their components and perhaps shed some light on their neurobiological role, we have developed a method for isolating SBs from rat brain. The protocol relies on a combination of centrifugational forces, sucrose gradient, and immunoisolation. Samples enriched in SBs were incubated with antibodies to HAP1B or to LR11 followed by incubation with FITC conjugated secondary antibodies. Anti-FITC coated beads were incubated with samples and SB-bead complexes formed were separated by magnetic sorting without pelleting the complexes during the isolation procedure. Immunopurified SBs, visualized by light and electron microscopy, show similar ultrastructure to those present in neurons.
Torre
Enrique R
ER
Department of Neurology, Emory University School of Medicine, Woodruff Memorial Research Building, 30322, Atlanta, GA, USA.
Coleman
Sarah
S
Yi
Hong
H
Gutekunst
Claire Anne
CA
D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D001923:Brain Chemistry / Q000502:physiology*; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D002479:Inclusion Bodies / Q000737:chemistry* / Q000648:ultrastructure; D026901:Membrane Transport Proteins; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011973:Receptors, LDL / Q000276:immunology / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry / Q000201:enzymology
D000906:Antibodies; C490757:Hap1 protein, rat; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011973:Receptors, LDL; C491167:Sorl1 protein, rat