Sarkosyl-insoluble fraction proteins were prepared from frozen samples of the hippocampus, hypothalamus, and midbrain periaqueductal gray matter of the patient, the brainstem of a previously reported anti-IgLON5 case (patient 7 described in Lancet Neurology)1 (link) fulfilling the postmortem diagnostic criteria of IgLON5 tauopathy, and the prefrontal cortex of a patient with Alzheimer disease (AD). Briefly, samples were homogenized and centrifuged twice at 20,000g. The supernatants were combined and incubated with 1% N-lauroylsarcosine (wt/vol) for 1 hour and centrifuged at 100,000g to get the sarkosyl-insoluble pellets that were subjected to immunoblot.
Immunoblots were run following denaturing standard procedures, proteins were transferred to a nitrocellulose membrane, and strips were incubated with anti-pTau (AT8, Thermo-Scientific, Rockford), anti-3Rtau (Merck-Millipore, Billerica, MA), and anti-4Rtau (Cosmo Bio, Tokyo, Japan) antibodies, appropriate secondary antibodies, and the results were visualized by enhanced chemiluminescence.
Immunoblots were run following denaturing standard procedures, proteins were transferred to a nitrocellulose membrane, and strips were incubated with anti-pTau (AT8, Thermo-Scientific, Rockford), anti-3Rtau (Merck-Millipore, Billerica, MA), and anti-4Rtau (Cosmo Bio, Tokyo, Japan) antibodies, appropriate secondary antibodies, and the results were visualized by enhanced chemiluminescence.
