β 3 tubulin tuj1

HiPSCs were differentiated inMNs according to the protocol described by Amoroso et al. [13 (link)]. At day 20, EBs were dissociated with PAPAIN (Worthington, Biochemical Corporation, Lakewood, NJ, USA) and plated onto poly-lysine/laminin-coated 8-well chamber slides (BD Biosciences, Two Oak Park, Bedford, MA, USA). Immunocytochemistry was carried out for the detection of specific neural markers: β-III-tubulin (TUJ1; Abcam, Cambridge, UK; 1:500), and LIM3 (Millipore Corporation, Billerica, MA, USA; 1:250). The cell nucleus was labeled with 4,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich, St. Louis, MO, USA) and examined under a fluorescence microscope. Images were acquired using a Zeiss (Thornwood, NY, USA) Axioplan 2 microscope.
All values provided in the text and figures are means of three independent experiments ± standard deviations (SD). Mean values were compared using the two-tailed Student t-test, for independent samples. p-Value was considered significant *** p < 0.001, ** p < 0.01, * p < 0.05. FACS Statistical analysis was performed according to paired Student’s t-test by using GraphPad Prism Software version 5.03 (GraphPad Software Inc., La Jolla, CA, USA).

Cell culture associated reagents were purchased from Gibco, Thermo Fisher: Dulbecco's Modified Eagle's Medium (DMEM), 0.25% Trypsin-EDTA solution. Penicillin/Streptomycin solution was from PAA, Cambridge, UK. Brain derived neurotrophic factor was obtained from Alomone Labs, Jerusalem, Israel. Recombinant human Aβ40 and Aβ42 peptides (TFA salts, purity >97%) were from rPeptide, Bogart, GA 30622, USA. Tween-20 (Ferak, Berlin, Germany) WST-1 cell viability assay was purchased from Roche, Switzerland, and the Caspase-Glo assay kit from Promega Co, Madison, WI, USA. 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), poly-L-lysine, retinoic acid, CalceinAM (calcein-acetoxymethyl ester), sodium chloride; goat serum; staurosporine, 4',6-diamidino-2-phenylindole (DAPI), PBS were obtained from Sigma Aldrich.
Antibodies against a microtubule component βIII-tubulin TUJ-1 were obtained from Abcam, Cambridge, UK; anti-APP/Aβ antibody—from Millipore, Darmstadt, Germany. Guinea pig anti-Synaptophysin1 was from Synaptic Systems, Goettingen, Germany. Secondary antibodies were Alexa-488 and Alexa-568 from Invitrogen.