Multimodal Analysis of Neuronal Markers

Mice were sacrificed and transcardially perfused with 4% paraformaldehyde (PFA) in cold phosphate-buffered saline (PBS). Mouse brains, superior cervical ganglions, and adrenal glands were collected, post-fixed in 4% PFA/PBS solution overnight, submerged in 30% sucrose in PBS for at least 72 h, and sectioned at 40 μm thickness using CM1950 cryostat (Leica)^{22 (link)}. Frozen sections were stained with antibodies specific to p150^Glued (amino acid 3–202 at the N-terminus of p150^Glued, BD Biosciences, #610474, 1:200, recognizing p150^Glued but not p135+), p150^Glued & p135+ (amino acid 1266–1278 at the C-terminus of p150^Glued, Abcam, #ab11806, 1:500, recognizing both p150^Glued and p135+), tyrosine hydroxylase (TH, Pel-Freez, #P40101-150, 1:2500; ImmunoStar, #22941, 1:500; Synaptic Systems, #213104, 1:500), dopamine transporter (DAT, Millipore, #MAB369, 1:500), vesicular monoamine transporter 2 (VMAT2, Synaptic Systems, #138302, 1:1000), glial fibrillary acidic protein (GFAP, Abcam, #ab7260, 1:1000), TAR DNA-binding protein 43 (TDP-43, Proteintech, #10782-2-AP, 1:500), α-synuclein (Santa Cruz, #sc-7011-R, 1:500; Santa Cruz, #sc-69977, 1:500), phosphorylated α-synuclein (Ser129) [p-α-synuclein (Ser129), Abcam, #ab51253, 1:500], neuronal nuclei (NeuN, Millipore, #ABN91, 1:500), synaptophysin (Millipore, #AB9272, 1:500), binding immunoglobulin protein (BiP, also referred to as GRP78, Abcam, #ab21685, 1:500), reticulon 3 (RTN3, Proteintech, #12055-2-AP, 1:500), 63 kDa cytoskeleton-linking membrane protein (CLIMP63, Proteintech, #16686-1-AP, 1:500), calnexin (Abcam, #ab22595, 1:500), protein disulfide isomerase (PDI, Proteintech, #11245-1-AP, 1:500), receptor binding cancer antigen expressed on SiSo cells (RCAS1, Cell Signaling Technology, #12290, 1:500), early endosome antigen 1 (EEA1, Cell Signaling Technology, #3288, 1:500), sequestosome 1 (SQSTM1, MBL, #PM066, 1:500), cathepsin D (R&D Systems, #AF1029, 1:500), ER-Golgi intermediate compartment 53 kDa protein (ERGIC53, Sigma-Aldrich, #E1031, 1:500), 130 kDa cis-Golgi matrix protein (GM130, BD Biosciences, #610822, 1:500), phosphorylated eukaryotic translation initiation factor 2α (Ser51) [p-eIF2α (Ser51), Abcam, #ab32157, 1:500], and phosphorylated inositol-requiring enzyme 1α (Ser724) [p-IRE1α (Ser724), Abcam, #ab48187, 1:500] as suggested by manufacturers. Alexa Fluor 488-, 546-, or 647-conjugated secondary antibody (Invitrogen, 1:500) was used to visualize the staining. Fluorescent images were captured using LSM 880 laser-scanning confocal microscope with Zen software (Zeiss) in conventional or Airyscan mode. As a high-resolution imaging modality, the Airyscan technology is reported to improve resolution 2-fold and signal-to-noise ratio 8-fold relative to the conventional confocal microscopy^{61 (link)}. The paired images in all the figures were collected at the same gain and offset settings. Post-collection processing was applied uniformly to all paired images. The images were presented as a single optic layer after acquisition in z-series stack scans at 1.0 μm intervals from individual fields or displayed as maximum-intensity projection or three-dimensional (3D) reconstruction to represent confocal stacks.