Alexa fluor 555 phalloidin

Cells were grown on coverslips, fixed with 4% (w/v) paraformaldehyde, blocked, and incubated with primary anti Ki67 antibody (D3B5,Cell Signaling technology, Danvers, MA, USA) used as a marker for proliferation and alpha-Smooth Muscle Actin alpha (α-SMA) (A2547,Sigma, St. Louis, MO, USA) as myofibroblasts differentiation marker. Primary antibodies were detected with Alexa-conjugated secondary antibodies (Invitrogen). F-actin was stained with Alexa-Fluor-555-phalloidin (Invitrogen Molecular Probes, Carlsbad, CA, USA) and DNA was stained with Hoechst (Sigma, St. Louis, MO, USA). Images were acquired with a spinning disk Olympus IX81 microscope.

Neurons were fixed at DIV 14 in cold methanol 100% for 10 min. Primary antibodies were applied in GDB buffer (30 mM phosphate buffer, pH 7.4, containing 0.2% gelatin, 0.5% Triton X-100, and 0.8 M NaCl), overnight at 4 °C. Primary antibodies used were: mouse anti-LRRK2 1:100 (clone N231B/34, NeuroMab), rabbit anti-β-3Cav2.1 1:50 (Sigma), rabbit anti-synapsin I 1:200 (Cell Signalling). Secondary antibodies were prepared in GDB buffer and used for 2 h at room temperature; secondary antibodies include: goat anti-mouse Alexa Fluor 488 (Invitrogen), goat anti-rabbit Cy3 (Jackson Immunoresearch) and Alexa Fluor 555 Phalloidin (Molecular Probes, Life Technologies). Cover slips were mounted with prolonged reagent (Life Technologies) and observed with Zeiss Observer Z1 microscope equipped with an Apotome module. The obtained images provide an axial resolution comparable to confocal microscopy^{44 (link),45 (link)}. Images were acquired with AxioObserv Z1 microscope equipped with Apotome module using a plan-Apochromat 63×/1.40 Oil objective, pixel size 0,102 μm × 0.102 μm. Colocalization studies were performed on the single plan generated by optical sectioning elaborated by Apotome module. Acquired images were analyzed with ImageJ software using the colocalization analysis plugin to detect colocalization between LRRK2 and interactors.

After 72 h of culture, cells were fixed in 4% PFA for 20 minutes at room temperature, washed in PBS, and permeabilized in 0.5% Triton X-100 in PBS for 30 minutes. Then, cells were blocked in 5% BSA IgG free and 0.1% Tween-20 in PBS for 1 h at room temperature and (i) for PPARγ expression, cells were incubated with primary PPARγ antibody (E-8 from Santa Cruz, 1-100) in blocking solution; (ii) for PPRE-H2B-eGFP transfected cells, only Alexa Fluor® 555 Phalloidin and DAPI labeling were performed. The next day, cells were rinsed three times with 0.1% Tween-20 in PBS (PBST), then the staining was revealed with VectaFluor™ Excel R.T.U. Antibody Kit, DyLight® 488, Anti-Mouse IgG (DK-2488, Vector Laboratories) according to the manufacturer's instructions. After three washes with PBST, cells were incubated with Alexa Fluor 555 Phalloidin (Molecular Probes, 1/200 in PBST) for 1 h, in the dark at room temperature, then washed three times in PBST, and counterstained with DAPI for 10 mins at room temperature. Finally slides were mounted with Fluoromount-G (Molecular Probes) and stored at 4°C. Confocal microscopy images (obtained with a Leica spinning-disk microscope equipped with a Plan Apo 63X/1.4 oil objective and a CoolSnap HQ2 CCD camera) were processed with ImageJ (National Institutes of Health, https://imagej.nih.gov/ij/) or Icy (Institut Pasteur, http://icy.bioimageanalysis.org/).

Anti-Akt (cat. 2938s) and anti-p-Akt (S473, cat. 4060s) antibodies were acquired from Cell Signaling (Danvers, MA, USA), anti-α-tubulin (sc-8035), -β-actin (cat. sc-47778), -GFP (cat. sc-833s), -GST (cat. sc-138), -HA (cat. sc-7392), -Id2 (cat. sc-398104, -489), and -Sirt2 (cat. sc-28298) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA), anti-FLAG (cat. F 1804), -Sirt2 (cat. S 8447), -acetylated tubulin (cat. T 7451), and -Tuj (cat. T 2200) antibodies from Sigma-Aldrich (St. Louis, MO, USA), anti-β-tubulin (cat. 801202) antibody from BioLegend (San Diego, CA, USA), and anti-NeuN (cat. ABN 78) antibody from Merck Millipore (Billerica, MA, USA). Alexa Fluor 555 Phalloidin, Alexa Fluor 546 goat anti-rabbit, and Alexa Fluor 488 goat anti-mouse secondary antibodies were obtained from Molecular Probes (Eugene, OR, USA). Anti-HSP70 was obtained from Abcam (Cambridge, MA, USA). The siRNA for silencing Id2 (5′-GAGCUUAUGUCGAAUGAUAUU-3′) and the siRNA for silencing Sirt2 (5′-AATCTCCACATCCGCAGGCAT-3′) were obtained from Genolution (Republic of Korea). Akt inhibitor VIII and nicotinamide adenine dinucleotide hydrate (NAD⁺) were obtained from Sigma (St. Louis, MO, USA).