Rabbit anti th

Under deep anesthesia, mice were perfused with saline and then with PFA (4%). Brains were collected and post‐fixed in 4% PFA at 4 °C for 24 h. Brains were then immersed in 20% sucrose for 2 days at 4 °C and then sectioned into 40 µm coronal slices with a freezing microtome (thermo Scientific, Cryostat Nx70, USA). Brain sections were incubated in 0.3% hydrogen peroxide and then overnight in a PBS solution containing 0.1% Triton X‐100 and rabbit anti c‐fos (1:10 000, Sigma Aldrich, USA), rabbit anti TH (1:1000, Sigma Aldrich, USA) or rabbit anti BMAL1 (1:500, ThermoFisher, USA) at 4 °C. Afterward, sections were washed three times (10 min each) in PBS. Sections were then incubated for 2 h in biotinylated antibody (1:1000, Jackson ImmunoResearch Laboratories). Sections were again washed with PBS solution three times (10 min each) and then treated with avidin‐biotin complex (1:1000, Vectastain ABC Elite kit, Vector laboratories) for 1 h. After another round of 3 successive washing by PBS (10 min each), staining was visualized by monitored reaction with 3,3′‐diaminobenzidine and 0.01% hydrogen peroxide. The reaction was subsequently stopped by rising sections four times with PBS (10 min each). Sections were then mounted on gelatinized slides, dried and dehydrated in increasing gradients of ethanol, cleared in toluene, and cover‐slipped with Depex.

We verified the specificity of GCaMP6f expression to dopaminergic structures by comparing direct eGFP fluorescence to immunoreactivity to tyrosine hydroxylase (TH-ir). Acute slices of midbrain and striatum were fixed overnight at 4 °C in 4% paraformaldehyde dissolved in PBS, then stored in PBS. After resectioning to 40 µm, free-floating sections were washed in PBS 5 × 5 min and incubated in 0.5% Triton X-100, 10% normal goat serum and 10% foetal bovine serum for 30 min. Slices were subsequently incubated overnight with 1:2000 primary (rabbit anti-TH; Sigma) antibody dissolved in PBS containing 0.5% Triton X-100, 1% normal goat serum and 1% foetal bovine serum. Sections were then washed with PBS 5 × 5 min and incubated for 2 h at room temperature with 1:1000 secondary (DyLight 594 goat anti-rabbit; Jackson) antibody dissolved in PBS containing 0.5% Triton X-100, 1% normal goat serum and 1% foetal bovine serum. Sections were washed with PBS and mounted on gelled slides with Vectashield mounting medium (Vector Labs) and imaged using a Zeiss LSM880 (confocal) running Zen black version 2.3, at 20 ×, N.A. 0.8. Maximum intensity projection from a z-stack of height 30 µm was captured individually and the stack of the pictures were compressed. TH (red) was captured at 638–759 nm with 633 nm excitation light. GCaMP (green) was excited with 488 nm and captured at 493–630 nm.

Mouse tissues were lysed in T-PER reagent with a protease inhibitor cocktail (Thermo Scientific). Equal amounts of proteins were fractionated by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes. Blots were probed overnight at 4°C with the following primary antibodies: rabbit anti-TH (1:4000, Sigma), mouse anti-β-actin (1:2000, Abgent), rabbit anti-GFAP (1:4000, Millipore), mouse anti-GAPDH (1:5000, Santa Cruz). Next, the membranes were incubated for 1 h with IRDye 680LT goat anti-mouse IgG (H+L) (926-68020, Li-Cor, USA) or IRDye 800CW goat anti-rabbit IgG (H+L) (926-32211, Li-Cor, USA) (1:10,000 dilutions in TBST with 0.02% SDS) and protein bands detected by an Odyssey infrared imaging system (Li-Cor, USA). Protein levels were quantified by densitometry using Quantity One 4.5.2 software (Bio-Rad, Hercules, USA).

For immunohistochemistry, the antigen recovery was carried out in citrate buffer (pH 6) using a microwave (3 cycles of 5 min). The endogenous peroxidase was blocked (0.3% H₂O₂), and tissue sections were incubated over night with the primary antibody (rabbit anti-TH: 1:500, Sigma-Aldrich^®, São Paulo, Brazil; rabbit anti-GFAP 1:50, Sigma-Aldrich^®, São Paulo, Brazil). Sections were processed by the avidin-biotin peroxidase system (1:125, Kit ABC, Vectastain, Vector Laboratories^® (São Paulo, Brazil), and immune-positive cells were visualized by addition of the chromogen 3,3-diaminobenzidine (DAB, 1 mg·mL⁻¹, Sigma-Aldrich^®, São Paulo, Brazil) and hydrogen peroxide (0.2%). The tissue was washed in phosphate-buffered saline between procedures. Immunopositive cells were revealed by a brown reaction product. The sections were dehydrated in ethanol, cleared in xylene and cover-slipped for optic microscopic observations. In all experiments, tissues from every group were always processed in the same assay [22 (link),23 (link)].

Brain slices were blocked for 1 h at room temperature with 5% bovine serum albumin, incubated with a mixture of mouse anti-α-syn (1:1000, BD Transduction Laboratories, Cat # 610787) and rabbit anti-AQP4 (1:300, Millipore, Cat # AB3594), rabbit anti-laminin (1:300, Sigma-Aldrich, Cat # L9393) or rabbit anti-TH (1:500; Sigma-Aldrich, Cat # SAB4502964), or mouse anti-CD31 (1:500, Sigma-Aldrich, Cat # P8590) and rabbit anti-AQP4. Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies (Thermo Fisher Scientific, 1:1000, Cat # A-11034; Cat # A-11032) were incubated for 1 h at room temperature. Meninges were incubated with a monoclonal mouse anti-lymphatic vessel endothelial hyaluronan receptor 1 (lyve-1) antibody (1:1000, Abcam, Cat # ab33682) and thereafter incubated with Alexa Fluor 488 secondary antibody.

P17 cerebellum or midbrain tissue was lysed in M-PER reagent using a protease inhibitor cocktail (Thermo Scientific). Nuclear proteins and plasma proteins were isolated using a commercial extraction kit (Sigma). Equal amounts of proteins were fractionated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with primary antibodies. The following primary antibodies were used: rabbit anti-TH (1:500, Sigma), mouse anti-β-catenin (1:2000, BD bioscience), rabbit anti-Lrp5 (1:100, Cell Signaling Pathway), rabbit anti-Lrp6 (1:500, Cell Signaling Pathway), and mouse anti-GAPDH (1:2000, Santa Cruz) or rabbit anti-Histone3.1 (1:1000, CMCTAG) was used as an internal control. After primary antibody incubation, the membranes were treated with an HRP-conjugated secondary antibody and exposed for chemiluminescent detection (Thermo Scientific). The protein levels were analyzed using Adobe Photoshop CS5 (Adobe Systems) and normalized relative to the internal control.