Nb300 109

The immunohistochemical reaction protocol was described in detail by Ferran, et al. [69 ]. A tyrosine hydroxylase antibody was used (TH; Novus, Cat# NB300-109 RRID: AB 10,077,691, dilution 1:1000). The primary antibody was incubated overnight at 4 °C. After washes, the sections were incubated with biotinylated goat anti-rabbit antibody (Vector Laboratories, CA; used at 1:200 dilution) followed by a streptavidin–peroxidase complex (Vectastain-ABC kit; Vector Laboratories; 0.001% dilution), applied for 1 h at room temperature. Peroxidase activity was developed with 0.03% 3,30-diaminobenzidine (Sigma; St Louis, MO), plus 0.003% hydrogen peroxidase. After immunohistochemical and hybridization labeling, the slides were washed several times in PBS, air-dried, and cover slipped with Cytoseal 60 (Thermo Scientific, Ref. 8310–16) or Mowiol (Calbiochem, Bad Soden, Germany, Ref. 475,904). We verified the specificity of the antibodies by performing parallel control experiments that omitted the primary antibody, checking that no residual immunostaining was detected (data not shown). Immunoreaction shown in Fig. 8A was obtained with a pan-distalless (DLX) polyclonal antibody whose specifications are described by Puelles et al. [40 (link)]; this figure corresponds to their Fig. 8.15E.

The brain sections were washed with PBS and incubated in PBS/0.1% Triton (T-PBS) for 30 min at 20 °C while shaking. After incubation, the sections were blocked in T-PBS, 20% horse serum (Gibco Invitrogen) for 30 min at 20 °C while shaking. Following blocking, brain sections were incubated with primary Aβ antibody (Aβ, 1–16 (6E10), Covance, 1:1000) in T-PBS and 0.2% bovine serum albumin (BSA) for 2–3 days at 4 °C. The sections where then washed and incubated with anti-mouse fluorescent Alexa 546 antibody (Invitrogen-Life tech, Vienna, Austria) in T-PBS and 0.2% BSA for 1 hour at 20 °C while shaking. Finally the sections were washed and then mounted onto glass slides and coverslipped with Mowiol^® 4–88 (Roth, Austria). Some sections were stained with Thiazine Red or Resorufin (1.6 μg/ml, Sigma, overnight) to label plaques or CAA respectively. Some sections were counterstained with tyrosine hydroxylase antibodies (Novus, NB300-109; 1:1000, secondary anti-rabbit Alexa 488). Some sections were counterstained with blue DAPI (1:10 000, 1 hour) to visualize nuclei.

1,25(OH)₂D₃ and 6-OHDA were purchased from Sigma-Aldrich (1,25(OH)₂D₃; 17936, 6-OHDA; H4381, Sigma-Aldrich, St. Louis, MO, USA). The following primary antibodies were used: rabbit antibody to tyrosine hydroxylase (TH) (NB300-109, 1:2000, Novus Biologicals, Littleton, CO, USA), rabbit antibody to GFAP (ab7260, 1:1000, Abcam, Cambridge, UK), rabbit antibody to CD31 (ab28364, 1:1000, Abcam, Cambridge, UK), mouse antibody to CD31 (#550274, 1:1000, BD biosciences, Franklin lakes, NJ, USA), rabbit antibody to VDR (ab3508, 1:1000, Abcam, Cambridge, UK), mouse antibody to P-gp (sc-55510, 1:1000, Santa Cruz, Dallas, TX, USA), mouse antibody to pS129-α-synuclein (#825701, Biolegend, San Diego, CA, USA), and mouse antibody to α-synuclein (BD-610787, BD Biosciences, Franklin lakes, NJ, USA).
The following secondary antibodies were used: biotin-conjugated goat antibody to rabbit IgG (cat# BA-1000, 1:1000, Vector Laboratories, Burlingame, CA, USA), Alexa fluor 568-conjugated donkey antibody to rabbit IgG (A10042, Invitrogen, Carlsbad, CA, USA), Alexa fluor 488-conjugated donkey antibody to mouse IgG (A21202, Invitrogen, Carlsbad, CA, USA), and Alexa Fluor 405-conjugated goat antibody to mouse IgG (A31553, Invitrogen).

Immunofluorescence staining of differentiated cells was performed as previously reported [2 (link)]: The primary antibodies used were anti-βIII-tubulin mouse monoclonal (1:500, MMS-435P, Tuj1 clone, Covance, Princeton, NJ, USA); anti-tyrosine-hydroxylase (TH) rabbit polyclonal (1:100, NB300-109, Novus Biologicals, Centennial, CO, USA); anti-dopamine polyclonal antibody (1:250, IS1005, ImmuSmol, Pessac, France) with a STAINperfect immnostaining kit A (SP A-1000, ImmuSmol); anti-dopamine-transporter (DAT) rabbit monoclonal antibody (1:250, ab184451, Abcam, Cambridge, UK); and anti-Nurr1 rabbit polyclonal (1:50, 10975-2-AP, Proteintech, Rosemont, IL, USA). The secondary antibodies were Alexa Fluor^® 568-conjugated goat anti-mouse (1:400, A11004, Molecular Probes, Eugene, OR, USA); Alexa Fluor^® 488-conjugated goat anti-rabbit (1:400, A11008, Molecular Probes); and Alexa Fluor^® 568-conjugated goat anti-rabbit (1:400, A21069, Molecular Probes). Counterstaining was performed with 4′, 6-diamino-2-phenylindole, dihydrochloride (DAPI) (SE196, DOJINDO, Kumamoto, Japan). Images were collected using an LSM 710 microscope System with ZEN software (Carl Zeiss, Oberkochen, Germany).

Mice were anesthetized with isoflurane and perfused with ice-cold PBS. Brains were fixed with ice-cold 4% (w/v) paraformaldehyde overnight and cryoprotected in 30% (w/v) sucrose (in PBS) overnight at 4 °C. Thirty-five μm coronal sections were probed with the primary antibodies in the blocking buffer (PBS with 10% (v/v) goat serum and 0.1% (v/v) Triton X-100) overnight at 4 °C and then incubated with the appropriate fluorescence-conjugated secondary antibodies at 25 °C for 1 h. Images were acquired using a confocal microscope (Leica, Wetzlar, Germany).
For TH counting, sections were probed with rabbit anti-TH (NB300-109, Novus Biologicals, Centennial, CO, USA) in the blocking buffer overnight at 4 °C and then incubated with biotin-SP-AffiniPure goat anti-rabbit IgG H+L (111-065-045, Jackson ImmunoResearch, Philadelphia, PA, USA) at 25 °C for 1 h. We used streptavidin-conjugated HRP (Vectastain ABC kit, PK4000, Vector Laboratories, Burlingame, CA, USA) and 3,3′-diaminobenzidine tablets (D4293, Sigma-Aldrich, St. Louis, MO, USA) to visualize TH-positive neurons. The counting of total numbers of TH-positive neurons in the SN was conducted via an Optical Fractionator probe of Stereo Investigator software (MicroBrightfield, Williston, VT, USA).

Paraffin-embedded sections were immunostained with anti-TH (dilution 1:5000; NB300-109; Novus Biologicals) or with an anti-BrdU antibody (dilution 1:10; catalog 551321; BD Biosciences), as described previously (25 (link)). Vegfa and Hif-2α mRNA transcripts were detected via in situ hybridization on sections from paraffin-embedded tissues using the manual RNAscope 2.5 HD BROWN assay (Advanced Cell Diagnostics). Samples were imaged using a DM 1000 LED microscope (Leica Biosystems). Stereological estimation of cell number, cell density, and carotid body volume was performed using ImageJ (NIH) on every fourth paraffin section (25 (link)). In situ hybridization probe signal was quantified using trainable Weka segmentation in FIJI ImageJ software (NIH), according to the manufacturer’s instructions.