Mouse anti gfap

The following antibodies were used for immunoblotting: Mouse anti-calbindin (Sigma), rabbit anti-EAAT2 (Cell Signaling Technology), mouse anti-NeuN (Millipore), Rabbit anti-GluR1 (Millipore), rabbit anti-EAAT4 (Abcam) and goat anti-Lcn2 (R&D Systems). Goat anti-IKK2 (human specific, detects only the transgene), rabbit anti-IKK1/2, rabbit anti-ERK2, rabbit anti-EAAT1, rabbit anti-EAAT3, rabbit anti-galectin 3 (Mac-2), mouse anti-GFAP and all corresponding HRP coupled secondary antibodies were obtained from Santa Cruz Biotechnologies.
Rabbit anti-Prosap1 was kindly provided by Prof. Tobias Böckers (Ulm, Germany).
Goat anti-IKK2, mouse anti-GFAP, mouse anti-NeuN and Mouse anti-calbindin were also used for immunofluorescence. Additionally, following antibodies were used for immunofluorescence: mouse anti-Aldh1l1 (Abcam), rabbit anti-RelA (Santa Cruz Biotechnologies), PE-labeled rat anti-CD11b (eBioscience), and rat anti-CD45, rat anti-CD8, rat anti CD4 (all from BD Biosciences), rabbit anti-Iba1 (Wako) and chicken anti-GFP (Abcam). Corresponding Alexa Fluor-conjugated secondary antibodies were obtained from Molecular Probes (Life Technologies).

Most tissue preparation and immunostainings were performed as described previously (21 (link)) For DAB stainings in this study, the following primary antibodies were used: mouse anti-tyrosine hydroxylase (TH) (1:1,000, Immunostar, Hudson, WI, USA), rabbit anti-vesicular monoamine transporter 2 (VMAT2) (1:4,000, Immunostar Hudson, WI USA), mouse anti-human WT α-syn (1:2,000, Santa Cruz, CA, USA), and chicken anti-GFP (1:20,000 Abcam, Cambridge, UK). The SNpc sections were given an initial antigen-retrieval incubation in Tris/EDTA (pH 9.0) at 80°C for 45 min when stained for TH.
Double immunofluorescence stainings were performed as described previously (21 (link)). The primary antibodies used were rabbit anti-GSTA4 (1:100 Antibodies-online GmbH, Aachen, Germany), mouse anti-Gfap (1:1,000, Santa Cruz, CA USA), chicken anti-IBA1 (1:500 Synaptic Systems, Göttingen, Germany), and mouse anti-NeuN (1:1,000 Millipore, Billerica, MA USA) and were incubated together at 4°C. To compare immunofluorescent stainings of midbrain and striatum for Gsta4 and Gfap at 3 and 8 weeks, stainings were performed in parallel and images were taken with the same settings. All images were captured at high-resolution with the confocal Leica SP8 microscope (Leica Microsystems, Wetzlar, Germany).

Brain sections were prepared for free-floating immunohistochemical staining. The sections were blocked with 5% normal donkey serum and then incubated for 48 h at 4 °C with the primary antibody to Mouse Anti-NeuN (1:200, Millipore, mab377), Mouse Anti-GFAP (1:200, Santa Cruz, sc-33673) and Mouse Anti-AT-8 (1:200, Thermo Fisher Scientific MN1020). The sections were then incubated for 2 h at room temperature with a corresponding Donkey Anti-Mouse Alexa fluor 594-conjugated secondary antibody (1:400, Invitrogen, A-21203). The sections were mounted and imaged on a confocal laser scanning microscope. Analysis of cell counting was performed by using ImageJ software.

All fluorescent immunohistochemical experiments were performed following a standard procedure. Sections were incubated with a blocking and permeabilization solution (PBS containing 0.3% Triton-100X and 2% serum) for 30 min at room temperature, followed by incubation with primary antibodies in the same solution for 1 h at room temperature followed by overnight incubation at 4°C. After thorough washing with PBS, sections were incubated with fluorochrome-conjugated secondary antibodies for 2 h at room temperature. All sections were again thoroughly washed in PBS and PB and subsequently mounted on slides. Slides were enclosed using vectashield containing DAPI and dried. The following antibodies were used Chicken anti GFP, mouse anti GFAP, mouse anti PSA-NCAM, Rabbit anti S100B, rabbit anti Ki67.
DAB-based immunohistochemistry was performed as described previously (Oomen et al., 2010 (link); Schouten et al., 2015 (link)) using the following antibodies: goat anti DCX (Santa Cruz, 1:500), rabbit anti Ki67 (Abcam, 1:500), mouse anti GFAP (Millipore, 1:1000). Nissl staining was performed using Cresyl violet, as previously described (Heinrich et al., 2006 (link)).