Dapi fluoromont g

Cultured neurons were treated with or without 3-AB (400 µM final, 3 h; Fig. 1A–G) or CX-5461 (200 nM final, 1 h and 3 h; Fig. 7A–I) and then fixed for 10 min in 4% PFA in PBS (pH 7.4). The cultures were washed with PBS 3x (10 min) and incubated in 1% SDS in PBS (10 min) to improve nuclear immuno-detection [19] (link). The cultures were then washed with PBS 4x (15 min), incubated in blocking solution (0.1% Triton X-100, 2% NGS in PBS) for 1 h and then incubated overnight with primary AB (polyclonal anti-fibrillarin AB (1∶1000); Cat # ab5821, Abcam) in blocking solution or blocking alone (NP control). The cultures were rinsed with PBS 4x (10 min) and then incubated with secondary AB (GAR 568) for 4 h in darkness. After secondary AB incubation, the cultures were rinsed with PBS 4x (10 min), rinsed 2x with distilled water (5 min each), and mounted with antifade (DAPI Fluoromont-G; Cat # 0100-20; Southern Biotech).

Treated (100 nM Act-D, 45 min) and untreated hippocampal slices were collected, fixed and microsectioned as described above. To expose the fibrillarin epitope, the 40 µm sections were treated with 10 mM sodium citrate buffer (pH 9.0) for 30 min at 80°C [17] (link), washed in PBS (15 min), transferred to quenching solution for 10 min and incubated in PBS with 0.7% Triton X-100 for 15 min. The slices were rinsed with PBS 3x (10 min), incubated in blocking solution (0.1% Triton X-100 with 4% NGS, in PBS) for 2 h and then incubated overnight in anti-fibrillarin polyclonal AB (1∶500; Cat # ab5821, Abcam) diluted in blocking solution or blocker alone (NP). Next, the slices were rinsed with PBS 3x (10 min) and incubated with secondary AB (Goat Anti-Rabbit Alexafluor 488; 1∶200 Molecular probes) for 4 hours before being rinsed again with PBS 3x (10 min), 1x with distilled water (5 min) and mounted with antifade (DAPI Fluoromont-G; Southern Biotech).

Wt C57Bl/6 mice (1–3 months old, female) were anesthetized with ketamin/xylazin and perfused with PBS. After incubation of brains with 30 % sucrose for 24 h at 4 °C, brain tissue was embedded in TissueTek®O.C.T (Sakura, Alphen a.d.R., Nehterland), frozen at −80 °C and cut in 12 μm sections using a cryostat. Sections were fixed with 2 % PFA for 30 min, permeabilized with 0,5 % saponine for 10 min and treated with 3 % H₂O₂ for 10 min, followed by blocking with Roti®-ImmunoBlock (Roth, Karlsruhe, Germany) for 1 h. Samples were co-immunostained with primary antibodies mouse-anti-α-synuclein (1:200, syn-1,BD,New Jersey, USA) and rabbit-anti-SOD1 (1:100, ADI-SOD100, Enzo life science, New York, USA) in Roti®-ImmunoBlock for 1 h 45 min at RT. After washing with PBS, sections were blocked with 5 % goat-serum in PBS for 15 min and incubated with fluorophore conjugated secondary antibodies (1:750, goat-anti-rabbit-Alexa546 and goat-anti-mouse-Alexa-488, both Life technology, Carlsbad, USA) in 5 % goat serum for 1 h. Then sections were washed with PBS, incubated with xylol for 2 min and 100 % ethanol for 3 min and coverslipped using DAPI Fluoromont®G (SouthernBioTech, Birmingham, USA). To avoid unspecific binding of secondary antibodies, sections were also single stained with either α-synuclein or SOD1 primary antibody and both secondary antibodies.

Cells seeded over sterile coverslips and grown in 10% FBS were fixed in 4% PFA. Cells were permeabilized with 0.2% Triton in PBS and blocked with 5% BSA, 0.1% Tween-20. Coverslips were incubated overnight with rabbit anti-P-Histone H3 (Ser10) (Millipore) to determine proliferation or with rabbit anti-cleaved caspase 3 (R&D) to determine apoptosis. Negative controls were incubated with an irrelevant rabbit IgG (data not shown). An anti-rabbit Alexa Fluor 488 (Invitrogen) was used as the secondary antibody. Coverslips were mounted with DAPI Fluoromont-G (Southern Biotech), and IF was detected with an Olympus BX61 Microscope. Ten fields were quantified per coverslip at 10×. The number of P-Histone H3 positive cells was manually counted and compared to the total number of cells (DAPI positive) for each experiment, which was quantified using an ImageJ macro developed in the CRG Advanced Light Microscopy Unit facility (CRG, Barcelona).