Guinea pig anti synaptophysin 1

Immunoblotting was performed as previously described [57 ] with minor modifications. Briefly, transfected HEK293 cells were directly lysed in the dish with 0.5% SDS in PBS, supplemented with complete protease inhibitor cocktail (Roche) and Benzonase Nuclease (Sigma). All protein samples were boiled in reducing SDS loading buffer and separated on Bolt Bis-Tris Plus gels (Thermo Fisher). After transfer to nitrocellulose membranes (Thermo Fisher), membranes were incubated with the following primary antibodies overnight: mouse anti-PSD-95 (clone K28/43, BioLegend), guinea pig anti-Synaptophysin 1 (Synaptic Systems), rabbit anti-C4 (in house, Biogen 931–946), Beta-Actin HRP-conjugate (clone AC-15, Sigma). HRP-conjugated secondary antibodies were from GE Healthcare. Immunoreactivity was detected on ChemiDoc XRS+ (Bio-Rad) and analyzed with Image Lab software. mC4 levels were quantified by normalizing to the loading control, vinculin.

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.