Ab125096

hiPSC-derived CMs were detached from the plate by incubating with 1 ml of TrypLE (Thermo Fisher Scientific, 12604013) for 5 min at 37°C. A double volume of PBS was added to stop the reaction, and the cells were passed through a 100 μm cell strainer. hiPSC-derived CMs were centrifuged at 200 g for 5 min, fixed with 1% PFA for 20 min and washed 3x with PBS. hiPSC-derived CMs were incubated with primary antibodies against mVenus (Biorbyt, orb334993, 1:800) and mCherry (Abcam, ab125096, 1:250) and anti-cardiac troponin T-APC (Miltenyi Biotec, 130-120-403, 1:50) in blocking buffer (PBS, 4% donkey serum, 0.1% Triton X-100 in PBS, 2 mM EDTA) for 2 h at RT. After washing, the cells were incubated with the secondary antibodies anti-goat Alexa Fluor® 488 (Jackson ImmunoResearch, 705-546-147, 1:500), anti-rabbit Alexa Fluor® 555 (Abcam, ab150062, 1:500) and Hoechst 33342 (Thermo Fisher Scientific, H21492) for 1 h at 4°C. Cells were then washed 3x with PBS and centrifuged at 200 g for 5 min. Finally, 5 million cells were resuspended in 500 μl of FACS buffer (PBS, 2% FBS, EDTA) and kept on ice until further analysis. Cells were analyzed on Amnis ImageStream-X MkII (Luminex, United States).

Commercially available antibodies against DNAJA2 (rabbit monoclonal), DNAJB1 (mouse monoclonal), V5 tag (mouse monoclonal), GAPDH (mouse monoclonal), Pgk1 (mouse monoclonal) and mCherry (mouse monoclonal) were obtained from Abcam (UK)(ab157216; RRID:AB_2650527, Enzo life sciences (NY, USA) (ADI-SPA-450-E; RRID:AB_10621843), Invitrogen (CA, USA)(R960-25; RRID:AB_2556564), Sigma (G8795; RRID:AB_1078991), Invitrogen (459250; RRID:AB_2532235) and Abcam (ab125096; RRID:AB_11133266), respectively. Anti-mouse Ydj1 (SMC-150; RRID:AB_2570364) and anti-rabbit Sis1 (COP-080051; RRID:AB_10709957) were obtained from StressMarq Biosciences Inc. (Canada), and Cosmo Bio Co. (Japan), respectively. Antibody against YFP (rabbit polyclonal; RRID: AB_2650530) was generated in the laboratory. Anti-DnaK (rabbit polyclonal; RRID:AB_2650528) and anti-human Hsp/Hsc70 (rabbit polyclonal; RRID:AB_2650529) antibodies were a kind gift from Matthias Mayer (University of Heidelberg).

Upon completion of experiments, rats were deeply anaesthetized and perfused intracardially with 4% PFA in order to examine the diffusion of the virus. The brains were dissected, stored overnight at 4 °C, and finally transferred to 30% sucrose. Coronal sections (50 μm) were cut on a cryostat and collected in PBS. Free-floating sections were incubated in a blocking solution (4% bovine serum albumin (BSA), 10% normal goat serum and 0.5% Triton X-100) for 1 h at RT. Then, they were incubated in primary monoclonal mouse antibody to mCherry (1: 500 dilution, Abcam, ab125096) in the blocking solution overnight at 4°. Subsequently, sections were washed with PBS and incubated for 1 h at RT with secondary fluorescent labels AlexaFluor-568-labeled anti-mouse antibody (1:400 dilution, Lifetechnologies, A11036) diluted in PBS for 1 h on a shaker at room temperature. Sections were washed in PBS, mounted with mounting media containing DAPI (Vector, H1200) and coverslipped.

Embryonic hippocampal and cortical neurons were cultured as described previously.²⁴ (link) Briefly, neurons were seeded at a density of 200,000 cells/well in 6-well tissue culture dishes (Falcon, Glendale, AZ, USA) precoated with poly-D-lysine (0.1 mg/mL; Sigma-Aldrich) and cultured in defined medium of Neurobasal, l-glutamine (2 mm), penicillin-streptomycin, and B27 supplement (1:50; all from Thermo Fisher Scientific). On day 3, adeno-associated viruses (AAVs) were transduced at a multiplicity of infection of 100,000. On day 7, cultures were rinsed with Dulbecco's phosphate-buffered saline (DPBS) and subsequently lysed in 500 µL RIPA with protease inhibitor cocktail followed by sonication. Lysate was clarified with centrifugation at 10,000 × g for 10 minutes. Samples were boiled for 5 minutes in SDS sample buffer and run on 4% to 20% TGX gels (Bio-Rad). Then, 0.2-µm nitrocellulose membranes were probed with rabbit anti-Kif5a (ab5628, 1:2000; Abcam, Cambridge, MA, USA), mouse anti-FLAG (F1804, 1:1000; Sigma-Aldrich), mouse anti-mCherry (ab125096, 1:5000; Abcam), or mouse anti-GFP (ab1218, 1:5000; Abcam) diluted in 5% milk with 0.2% Tween-20 in tris-buffered saline (TBST).

Use 6% SDS-PAGE gel, and the separated proteins were transferred by electro blotting to NC membranes. The membranes were blocked with 5% non-fat dry milk in TBST and incubated with the primary antibody including anti-GFP antibody (Abcam, ab6658) and anti-mCherry antibody (Abcam, ab125096) overnight at 4 °C. Then washing three times, the second antibody including anti-Goat HRP (Proteintech, SA00001-3) and anti-mouse HRP (Proteintech, SA00001-8) for about 2 h Immunolabelling was detected using SuperSignal West Femto (Thermo Fisher Scientific, 34096).