Mouse anti th

Seven days post-surgery, animals (n = 27) were deeply anaesthetized (zoletil mixture; 0.1 ml/10 g; intraperitoneally) and perfusion-fixed with 4% formaldehyde. Brains were dissected, post-fixed, cryo-protected and coronally sectioned into 40 μm. Sections through striatum and midbrain were stained with hematoxylin/eosin for histological analysis (S1 Fig). Successive sections through the SN were immunostained with mouse anti-TH (1:1000, Chemicon) followed by incubation with Streptavidin-Biotinylated horseradish peroxidase complex (1:100; GE Healthcare). Unbiased stereological counting of TH-positive neurons was performed bilaterally in the SNpc, SNpr, and VTA of Aqp9^-/- and WT mice injected with MPP⁺ (Aqp9^-/-, n = 8; WT, n = 7) or saline (Aqp9^-/-, n = 3; WT, n = 3) as described elsewhere [42 (link),43 (link)]. A detailed description of the stereological counting procedure is provided in S1 File.

For immunohistochemistry(IHC), mice were euthanized then perfused transcardially with PBS, followed by PBS with 4% paraformaldehyde. Brains were dissected, cryoprocted with 30% sucrose in PBS then processed MultiBrain Technology (NSA, NeuroScience Associates, Knoxville, TN) for DAB IHC with a 1:75,000 dilution of Goat anti-EGFP serum according to the Vectastain elite protocol (Vector Labs, Burlingame, CA). Serial sections were digitized with a Zeiss Axioskop2 microscope at 10× magnification.
For immunofluorescent studies, brains were prepared as above, frozen and sectioned to 40 uM on a crytostat, and stored in PBS with 0.1% azide until use. Sections were blocked with 5% normal donkey serum and 0.25% triton and then incubated with Chicken anti-GFP(Abcam) and Mouse anti-Th (Chemicon) followed by appropriate Alexa dye-conjugated secondary antibodies (Invitrogen, Carlsbad, CA). Images were acquired as Z stacks (2 μm sections) with a Zeiss Inverted LSM 510 confocal microscope.
For Allen Brain Atlas images of Fig. 3, we selected for presentation the first 9 available coronal in situ hybridization image sets, alphabetically, from those transcripts with > 10 fold enrichment, p < 0.01, and pSI < 0.10e-6 (Table 1).

Half-brains were fixed overnight in 4% PFA at 4°C, dehydrated in 30% sucrose overnight, and sectioned on a freezing stage microtome into 30 μm thick coronal slices stored in cryoprotectant solution. For immunofluorescence, sections were washed extensively in PBS and blocked with 10% bovine serum albumin (BSA, Sigma, A2153) for 1 h at room temperature (RT).
Sections were immunolabeled for amyloid-beta (Aβ), microglia (Iba1), astrocytes (GFAP), tyrosine hydroxylase (TH), and a widely used marker for neuritic damage LAMP1^{15 (link),69 (link)–71 (link)}; in different combinations specified in appropriate figure legends. The following primary antibodies were used: biotin anti-Aβ (clone 6E10, 1:3000, BioLegend), rabbit anti-Iba1 (1:2000, Wako), guinea pig anti-GFAP (1:3,000, Synaptic Systems), mouse anti-TH (1:500, Millipore Sigma) and rat anti-LAMP1 (1:2000, Abcam). Sections were incubated in primary antibodies for 48 h at 4°C. The sections were washed with PBS and incubated in fluorescently labeled secondary antibodies/reagents (Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 594, streptavidin conjugate 594 and Alexa Fluor 647, Invitrogen; all at 1:1000) for 4 hr at RT, then mounted and coverslipped (Prolong Diamond, ThermoFisher Scientific).

For double labelling of orexin and tyrosine hydroxylase (TH), tissues were washed and incubated with a PBS cocktail consisting of 2% NDS, 0.3% Triton X-100, goat anti-orexin (1:100 of Cat #Sc-8070 Santa Cruz Biotechnology Inc.) and mouse anti-TH (TH: 1:1000; Cat #T1299, Sigma-Aldrich) antibodies at 4°C for 48 h. The sections were then washed and incubated in Alexa Fluor 594 donkey anti-goat secondary antibody (1:100; Jackson ImmunoResearch Laboratories Inc.) in 0.1M PBS for 2½ h. After washing in PBS, sections were incubated with Alexa-Fluor 488 donkey anti-mouse secondary antibody (1:100, Jackson) for 2½ h. Finally, the sections were rinsed in PBS and cover-slipped using Vecta Shield (Vector Laboratories) anti-fade mounting media.
Controls for each experiment were performed to determine whether the primary or the secondary antibodies produced false-positive results. The controls involved omission of the primary and/or secondary antisera to eliminate the corresponding specific labelling. Nonspecific activation of c-Fos was assessed by evaluating the CNS expression of c-Fos in animals receiving IP injection of PS.