Ab104225

Antibodies were purchased as follows: Primary antibodies: rabbit-anti-UCH-L1 (AB1761,MILLIPORE), rabbit-anti-synaptopodin (S9442, Sigma-Aldrich), mouse-anti-α-actinin4 (MAB1682, MILLIPORE), mouse-anti-nestin (MAB2763, R&D), rabbit-anti-NeuN (ab104225, Abcam), mouse-anti-S100 (ab14849, Abcam), rabbit-anti-NSE (ab53025, Abcam), mouse-anti-nephrin (sc-166574, Santa Cruz Biotechnology), mouse-anti-WT-1 (ab96792, Abcam). Secondary antibodies: peroxidase-conjugated goat-anti-mouse IgG, peroxidase-conjugated goat-anti-rabbit IgG, fluorescein (FITC)-conjugated goat-anti-rabbit/mouse IgG, cy3-conjugated goat-anti-mouse/rabbit IgG all from Protein Tech Group Inc., Chicago, USA, HRP-goat-anti-mouse IgG, HRP-goat-anti-rabbit IgG from Long Island Biotech Co., LTD, Shanghai, China.

Sections were washed and blocked for 1 h in 10% of host goat serum. Sections were incubated overnight at 4°C in a solution containing the antibody, NeuN rabbit (ab104225, 1:1,000, Abcam). After additional washes, the sections were incubated for 1 h at room temperature in a secondary solution containing the fluorescent markers, goat anti-rabbit 488 (A11034, 1:1,000, Invitrogen). After one additional wash, the sections were mounted on charged slides, counterstained, and coverslipped with Prolong Gold Antifade Mountant with DAPI.

Neurons were fixed with 2% (wt/vol) paraformaldehyde in PBS + 5% (wt/vol) sucrose. Cell membranes were permeabilized with 0.2% Triton-X in PBS. Cells were consecutively stained with mouse monoclonal antibody against phosphorylated neurofilament H, SMI-31 (1:1000; NE1022, Calbiochem), and AF-conjugated anti-mouse secondary antibody (Molecular Probes, Invitrogen). When indicated, neuronal cells were detected by staining against Neuronal Nuclei (NeuN) with rabbit polyclonal antibody (1:500; ab104225, Abcam) and AF-conjugated anti-rabbit secondary antibody (Molecular Probes). Nuclei were stained with DAPI (1:10,000; Molecular Probes). Devices processed for immunofluorescence underwent a final wash with PBS and were imaged in solution. Non-compartmentalized neurons, cultured on coverslips, were mounted onto slides with ProLong Diamond (Molecular Probes).

Autoclaved paraffin sections were incubated with a blocking solution containing 5% skim milk in TBST (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 1 h. Sections were incubated with the primary antibody (anti-p-syn antibody (mouse monoclonal, pSyn #64, 1:300, FUJIFILM Wako), neuronal marker (rabbit polyclonal, anti-NeuN, 1:500, ab104225, Abcam), oligodendrocyte marker (rabbit polyclonal, anti-GST-pi, 1:500, 312, MBL)) in TBST overnight at 4 °C, followed by incubation with the secondary antibody biotinylated anti-rabbit IgG (1:300, BA1000, Vector Labs) and biotinylated anti-mouse IgG (1:300, BA9200, Vector Labs). For diaminobenzidine staining, sections were quenched with 3% H₂O₂/methanol for 30 min before blocking and incubated with the VECTASTAIN Elite ABC Kit reagent (Vector Labs) for 30 min after secondary antibody incubation. Color development was performed using 3,3-diaminobenzidine/H₂O₂. To analyze the density of aggregation (p-syn) inclusions, whole-brain sections were imaged with a Keyence microscope using bright-field capture. Multiple fields were captured using a ×10 objective and stitched together using the Keyence Merge function. The density of α-synuclein aggregates was quantified using Hybrid Cell Count software based on hue.

All the immunohistological analyses were performed on the brain region displaying the coordinates: Bregma 0.00 mm. In this region, the ischemic core (striatum) is separated from the ischemic penumbra (cortex) by the corpus callosum, a structure that can be easily identified and allows differentiating the two zones (Supplementary Fig. 2B,C). Immunostainings were performed on brain sections of 20-μm using rabbit polyclonal IgG anti-NeuN, (Abcam ab104225, 1:500 dilution from original unit), goat polyclonal IgG anti-Iba1 (Abcam ab5076, 1:500 from original unit), rabbit polyclonal IgG anti-GFAP (Abcam ab7260, 1:500 dilution from original unit) as detailed in Supplementary Methods. For each animal the quantifications were performed on two different pictures of 90000 µm² (2 pictures taken within the striatum and 2 pictures taken within the cortex). Each picture has been gridded and 3 fields of 10000 µm² have been randomly selected. The number of positive cells per field was counted in the cortex and in the striatum. Data are presented as a number of positive cells/field. Quantifications were performed using the Histolab software (version 6.0.5, Microvision Instruments-Evry).

Chromogenic immunohistochemistry was performed on slide-mounted sections. Frozen sections were thawed by incubating at 37 °C for 15 min. A hydrophobic barrier was created surrounding each series of sections using an ImmEdge hydrophobic barrier PAP pen (Vector Laboratories, Burlingame, CA, USA). Sections were incubated overnight in the following primary antibodies: rabbit anti-Neuronal nuclear protein IgG (NeuN; Abcam, ab104225, 1:1500), rabbit anti-Ionized calcium binding adaptor molecule 1 IgG (Iba1; Wako, 019–19,741, 1:1500) or rabbit anti-Glial fibrillary acidic protein IgG (GFAP; Abcam, ab7260, 1:1500). Sections targeting NeuN underwent heat-induced antigen retrieval (15 min in 95 °C citrate buffer, pH 6.0) prior to primary incubation. Primary antibodies were detected by incubation with biotin-conjugated Donkey anti-Rabbit IgG secondary antibody (Jackson ImmunoResearch, 711-065-152, 1:400), followed by streptavidin–horseradish peroxidase complex (ABC elite kit, Vectastain) and ultimately exposed to di-amino-benzydine (DAB; Sigma-Aldrich, D8001). Sections were then rinsed three times in PBS (5 min each) and dehydrated in 50%, 70%, 95% and 100% ethanol solutions (30 s each). Finally, sections were cover slipped using DPX Mountant (Sigma-Aldrich) and left to dry at room temperature overnight.

The mouse antibodies used were as follows: anti-NeuN (Ab104225, Abcam, dilution IF: 1:500). Rabbit antibodies used in this study were as follows: anti-NRXN1 antibody was used (GTX54845, GeneTex, dilution, IF:1:30, IHC:1:30; Santa Cruz, sc136001, WB:1:100), anti-MAP 2 (Ab32454, Abcam, dilution IF: 1:500), anti-GFP (AF5066, Beyotime, dilution IF: 1:100), and anti-GAPDH (10494, Proteintech, dilution WB 1:1000). Other antibodies were as follows: Donkey polyclonal Secondary Antibody to Mouse IgG (Alexa Fluor594, ab150108, Abcam, dilution IF 1:1000), Donkey polyclonal Secondary Antibody to Rabbit IgG (Alexa Fluor488, ab150061, Abcam), HRP-conjugated AffiniPure Goat Anti-Mouse IgG(H+L) (SA00001-1, Proteintech, dilution WB:1:5000), and HRP-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) (SA00001-2, Proteintech, dilution WB:1:5000).