Fig. 4: PSER129 associates with PK-resistant alpha-synuclein in the mouse OB.

a Summary of approach for measuring PK resistance in situ. Step 1, the location of PSER129 is labeled using TSA. CF-568 dye is covalently bound to tissue (Position “B”). Step 2, low pH and heat are used to remove the antibody complex from the tissue while leaving the CF-568 dye intact. Step 3, tissues are briefly treated with PK to destroy enzyme-accessible epitomes of alpha-synuclein. Step 4, an antibody against total alpha-synuclein is used for TSA labeling of the remaining alpha-synuclein in the sample. (Position “A”). Results allow simultaneous visualization of PK-sensitive and PK-resistant epitopes. b PD brain sections processed with this dual-labeling protocol. High-magnification confocal images of the substantia nigra. The top panels depict results without PK and below with PK digestion (“PK”). c Low magnification confocal images of OB dual-labeled for PSER129 and alpha-synuclein. The top panels show an OB section processed in the absence of PK. The bottom panels depict OB sections processed with PK. d High-magnification images of the mitral cell layer. Expanded high-magnification images depicting a mitral cell body (e) and apical dendrite (f) in the EPL with (bottom panel) and without PK (top panel). g Distribution of PSER129 and PK-resistant alpha-synuclein in a single OB mitral cell. MCL mitral cell layer, EPL external plexiform layer. (WT mice n = 3, PD substantia nigra = 3).