Lsm 510 meta nlo multiphoton confocal microscope

Rabbit anti T22 (EMD Millipore CA, USA) and rabbit anti tau pThr²³¹ (Thermo Fischer) were used to probe tau inclusions for the presence of oligomeric tau (T22) and for phosphorylation at Thr²³¹. Tau oligomeric species are currently hypothesized to be more toxic to neurons than the fibrillar inclusions themselves [23 (link)], and pThr²³¹ is thought to be a key site in the regulation of tau protein folding and ability to interact with microtubules [3 (link), 24 (link)]. Double labeling was performed on hippocampus from one case each from AD, ALSci, FTD, MSA, DLDB, and mDLDB. Tau protein was probed for pThr¹⁷⁵ using rabbit primary antibody (1:1000) overnight at 4 °C and Alexa Fluor goat anti-rabbit 488 nm secondary (1:200, Thermo Fischer) for 1 h at room temperature. Rabbit anti tau pThr²³¹ antibody was then labeled using a Zenon primary antibody labeling kit with Alexa Fluor 555 nm dye (Thermo Fisher) and probed for 1 h at room temperature. Slides were stored overnight at 4 °C and visualized within 24 h of labeling by confocal imaging on a Zeiss LSM 510 Meta NLO multiphoton confocal microscope.