Alexa fluor 488 anti rabbit igg

Cells were grown as spheres in 96-well low attachment plates for 5 to 7 days before fixation with 4% paraformaldehyde for 20 min at room temperature. Spheres were permeabilised with 0.5% Triton X-100, blocked for 30 min with 0.5% BSA and 0.1% FBS in PBS with 0.1% Triton X-100, and stained with the following primary antibodies: rabbit anti-K14 (Poly19053); rat anti-K8 (TROMA-I, DSHB); and rabbit anti-SMA-Alexa-555 (Ab202509); followed by anti-rabbit IgG Alexa Fluor 488 (Thermo Fisher Scientific, A21206) and anti-mouse IgG Alexa Fluor 555 (Thermo Fisher Scientific, A21203) secondary antibodies and DAPI. All primary and secondary antibodies were used at a 1:500 dilution. DAPI was used at a 1:10,000 dilution. Spheres were mounted in Vectashield (Vector Laboratories) and visualised using 20× magnification on a Leica confocal SP8 microscope. Z projection with maximum intensity was used and quantification was performed using the integrated pixel intensity function in ImageJ.

Antibodies were used for both Western blots and immunofluorescence. Primary antibodies for Arp2, Arp3, α-tubulin, β-actin, E-cadherin, KinomeView Profiling kit, N-WASP, pan-actin, phospho-PLK1 (Thr210), PLK1, phospho-Rac1/Cdc42(Ser71), Rac1/Cdc42, vimentin, and WAVE-2 were purchased from Cell Signaling Technology (#3128, #4738, #2144, #4970, #3195, #9812, #4848, #8456, #9062, #4513, #2461, #4651, #5741, and #3659). PLK1 primary antibody used for immunofluorescence was purchased from Thermo Fisher Scientific (#37-7100); γ-actin antibody was purchased from Abcam (#ab123034). HCV NS5A sheep polyclonal antibody and NS5A mouse antibody were gifts from Professor Mark Harris (University of Leeds, UK) and Associate Professor Michael Beard (University of Adelaide), respectively. Secondary antibodies were either conjugated with HRP; anti-rabbit Ig/HRP (#P0448), and anti-mouse Ig/HRP (#P0161) (DAKO) or labelled with fluorescence dyes: antirabbit IgG Alexa Fluor 488 (#A21206), anti-mouse IgG Alexa Fluor 488 (#A21202), anti-mouse IgG Alexa Fluor 594 (#A21203), and anti-rat IgG Alexa Fluor 647 (#A21472) (Thermo Fisher Scientific).

Cells were cultured on coverslips coated with poly-d-lysine (Sigma-Aldrich, P7280) and incubated in standard conditions. Cells on coverslips were fixed with ice-cold methanol at 4 °C for 30 min. Cells were washed with PBS multiple times and incubated with 5% BSA (Sigma-Aldrich, A7906) at room temperature for 30 min. Next coverslips were incubated with primary antibodies at the indicated concentrations against pericentrin (0.1 μg ml^–1; Abcam, ab4448) and α-tubulin (0.5 μg ml^–1; Sigma-Aldrich, T6074) diluted in 1% BSA for 1 h at room temperature in a humidified chamber. Coverslips were washed with PBS and incubated with the fluorescent secondary antibodies anti-mouse IgG-Alexa Fluor 594 (2 μg ml^–1; Thermo Fisher Scientific, A-11005) and anti-rabbit IgG-Alexa Fluor 488 (2 μg ml^–1; Thermo Fisher Scientific, A-11034) diluted in 1% BSA for 1 h at room temperature in the dark. Coverslips were washed in PBS followed by mounting and counterstaining with DAPI with ProLong Diamond antifade mountant. Cell images were captured at ×63 resolution on a Zeiss Axioplan upright microscope. Images were analysed using Fiji (v.2.9.0)^{53 (link)}.

To characterize iPSC-derived neuronal cultures, neurons were fixed with 3.7% FA in 4% sucrose 14 days after the start of differentiation (d14). After blocking and permeabilization, neurons were stained with primary and secondary antibodies as described before [41 ]. The following antibodies were used: anti-TAU (rabbit, K9JA, Dako (Jena, Germany), A0024), anti-MAP2 (chicken, Abcam (Camebridge,. UK), ab5392), anti-vGlut1 (mouse, Merck Millipore (Burlington, MA, USA), MAB5502), anti-ChAT (rabbit, Thermofisher Scientific, PA5-26597), anti-SYP (mouse, Santa Cruz Biotechnology (Dallas, TX, USA), sc-17750), anti-vGlut1 (rabbit, Synaptic Systems (Göttingen, Germany), 135303), anti-PSD95 (mouse, Biolegend (San Diego, CA, USA), MMS-5182), anti-rabbit IgG Alexa-Fluor-488 (donkey, Thermofisher Scientific, A21206), anti-mouse IgG Alexa-Fluor-488 (donkey, Thermofisher Scientific, A21202), anti-chicken IgG AlexaFluor-647 (goat, Thermofisher Scientific, A21449), anti-rabbit IgG AlexaFluor-568 (goat, Thermofisher Scientific, A11011), anti-mouse IgG AlexaFluor-568 (donkey, Thermofisher Scientific, A10037).

The cells were fixed with ice‐cold methanol for 15 min for staining γ‐H2AX and with 4% paraformaldehyde for staining mitotic cells and YAP/TAZ, respectively. The fixed cells were permeabilised with 0.3% Triton X‐100 in PBS and blocked with 3% fetal bovine serum in PBS for 30 min. They were incubated with 1/1000 rabbit anti‐γ‐H2AX (Genetex, Irvine, CA, USA; GTX127340), 1/2000 mouse anti‐α‐tubulin (Sigma‐Aldrich; B‐5‐1‐2), 1/500 anti‐phospho‐Histone H3 S10 (Cell Signaling Technology; #9701) or 1/300 rabbit anti‐YAP/TAZ antibody (Cell Signaling Technology; #8418) at 4 °C overnight and then washed with PBS three times. Next, the cells were incubated with 1/500 anti‐rabbit IgG‐Alexa Fluor 488, 1/500 anti‐mouse IgG‐Alexa Fluor 488, anti‐rabbit IgG‐Alexa Fluor 594 or phalloidin‐Alexa Fluor 594 (Thermo Fisher) for 1 h at room temperature and again washed with PBS three times. Then, the cells were mounted in Prolong Diamond reagent containing 10 μg·mL⁻¹ of Hoechst 33342 (Thermo Fisher Scientific). Images were obtained with an FV1000‐D confocal microscope using fv10‐asw software (Olympus, Tokyo, Japan). To capture mitotic cells, ~ 40 images were collected with a z‐optical spacing of 0.2 μm with a 100× numerical aperture 1.4 objective lens. For other cells, single sections were collected with a 40x objective lens.