Guinea pig anti synapsin1 2

Pcdh19 antibody was raised by immunizing a rabbit with the GST-fused cytoplasmic region of Pcdh19 (Val1041-Leu1140 a.a.) and subsequently affinity-purified with the antigen. A GST-reactive population was also removed. The following antibodies were purchased: rat anti-Ctip2, chicken anti-LacZ and chicken anti-GFP (Abcam); mouse anti-Homer1 and guinea pig anti-Synapsin1/2 (Synaptic systems); rabbit anti-MAP2, mouse anti-Tau1 and guinea pig anti-Vglut2 (Millipore); rabbit anti-FLAG and mouse anti-MAP2 (Sigma-Aldrich); and mouse anti-Abi-1 and rabbit anti-RFP (MBL).

H32 neurospheroids were stained by immunohistochemistry (IHC). Briefly, neurospheroids were washed with PBS and fixated using 4% paraformaldehyde or absolute methanol, depending on the primary antibody. Following additional washing steps, spheroids were blocked in 10% normal goat serum or 1% bovine serum albumin containing 0.3% Triton X100. Then, neurospheroids were incubated overnight at 4 °C with primary antibodies as follows: chicken anti-MAP2 (#188006, Synaptic Systems, Göttingen, Germany), guinea pig anti-Synapsin 1/2 (#106004, Synaptic Systems), guinea pig anti-Tau (#314004, Synaptic Systems), mouse anti-beta actin (#3700s, Cell Signaling, Danvers, MA, USA) or goat anti-Integrin beta1 (#sc6622, Santa Cruz, Dallas, TX, USA). After 24h, spheroids were washed with PBS and incubated with the compatible secondary antibody: goat anti-chicken Alexa Fluor 488 (#A11039, Invitrogen, Carlsbad, CA, USA), goat anti-guinea pig Alexa Fluor 488 (#A11073, Invitrogen), goat anti-mouse Alexa Fluor 488 (#A11001, Invitrogen), or rabbit anti-goat Alexa Fluor 488 (#A11078, Invitrogen). Spheroids were also incubated with Phalloidin Alexa Fluor 488 (#8878, Cell Signaling) or Phalloidin iFluor 594 (#ab176757, Abcam, Cambridge, UK) diluted in blocking solution for 1 h. Cell nuclei were counterstained with Hoechst 33,342 (#H1399, Thermo Fisher, Waltham, MA, USA).