Rabbit anti ubiquitin

Cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS), permeabilized with 100% methanol, washed with PBS, and immunostained overnight at 4°C with the following primary antibodies at 1:200 dilutions; mouse monoclonal TuJ1 (R&D systems, Minneapolis, MN, USA), rabbit anti-phosphoTDP-43 (pSer409/410) (Cosmo Bio), rabbit anti-TDP-43 C-terminus (Abcam, Cambridge, MA, USA), rabbit anti-GFP (Abcam), rabbit anti-GFAP (DAKO, Glostrup, Denmark), rabbit anti-ubiquitin (DAKO) and rabbit anti-CNP (Cell Signaling Technology, Danvers, MA, USA). The cells were then incubated with Alexa Fluor 488- or 633-conjugated goat anti-rabbit or anti-mouse antibodies (Thermo Fisher) at 1:400 dilutions for 45 min at room temperature, followed by incubation for 15 min with 2 μg/mL Hoechst 33342 (Thermo Fisher). After washings, coverslips were mounted on glass slides with Gelvatol (20% glycerol/10% polyvinyl alcohol in 0.1 M Tris-HCl pH 8.0). Immunostained cells were examined under an AX80TR microscope equipped with a DP70 CCD camera (Olympus) or a TCS SP8 confocal microscope (Leica, Wetzlar, Hessen, Germany).

The absolute number of extracellular NM aggregates was estimated within the same sections in which SNpc TH-positive stereological cell counts were performed (i.e. serial 5-µm-thick paraffin-embedded sections covering the entire SNpc, taking every 17th section in rats and every 6th section in mice, for a total of 6–8 sections analyzed/animal). The number of neuronophagic events were assessed in serial H&E-stained 5-µm-thick paraffin-embedded sections covering the entire SNpc. The number of p62-immunopositive Marinesco bodies, PB, and Lewy body-like inclusions was counted from SNpc sections fluorescently immunostained with guinea pig anti-p62 (1:1000, Progen), rabbit anti-ubiquitin (1:500, Dako), and mouse anti-aSyn (1:1000, BD Biosciences). The total number of p62-positive inclusions falling into each category was counted from images covering the whole SNpc region in each section. Quantifications were performed in AAV-hTyr-injected at different time-points post-AAV-hTyr injection: 0.5 m (n = 8), 1 m (n = 5), 2 m (n = 6), 4 m (n = 5), 12 m (n = 6), and 24 m (n = 5). AAV-hTyr-injected mice were analyzed at 2 m post-AAV injections: WT (n = 3), aSyn KO (n = 4). AAV-hTyr (n = 5), AAV-TFEB (n = 4), AAV-hTyr + TFEB (n = 4), and vehicle-injected rats (n = 7) were analyzed at 12 m post-injection. All quantifications were performed by an investigator blinded to the experimental groups.

Immunolabeling of fixed guts were carried out as before^{42 ,50 (link)}. Primary antibodies used in this study were rat anti-mCherry (1:300), rabbit anti-p62 (1:2,000)^{31 (link)}, chicken anti-GFP (1:1,500, Invitrogen, A-10262), mouse anti-Delta (1:100, DSHB, C594.9B), rabbit anti-phospho-histone H3 (1:300, Millipore, 06–570), mouse anti-Prospero (1:100, DSHB, MR1A), rabbit anti-Pdm1 (1:50, gift of Fernando Jimenez Diaz-Benjumea), rabbit anti-ubiquitin (1:500, DAKO), mouse anti-H2Av (1:50, DSHB, UNC93-5.2.1), rabbit anti-Cdc2 (1:200, Santa Cruz Biotechnology, sc-53), rabbit anti-Cleaved Caspase 3 (1:50, Cell Signaling, 9661), rat anti-Atg8a (1:300)^{51 (link)} and secondary antibodies Alexa Fluor 488 anti-chicken A11039, Alexa Fluor 568 anti-rabbit A11011, Alexa Fluor 568 anti-mouse A11004 (all 1:1,500; Invitrogen). All stainings were repeated at least once, with similar results.