A21450

After co-culturing of iNeurons and astrocytes for 38 days on chips and well plate, cells were washed with ice cold phosphate buffered saline (PBS, Gibco), fixated for 15 min at room temperature with 4% formaldehyde (Thermo scientific) and subsequently washed 3 times with PBS. Cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) solution for 10 min at room temperature. 5% goat serum (Sigma Aldrich) in PBS was used as a blocking agent for 1 h at room temperature. Primary antibodies (MAP2, Rabbit polyclonal, Abcam ab32454, 1:1000; Synapsin-1/2, Guinea Pig, Synaptic systems 106004, 1:500) were diluted in PBS with 1% goat serum and applied to the cells, which was then incubated overnight at 4 °C. Cells were washed 10 times for 1 min with PBS. Secondary antibodies (Chicken anti-Rabbit IgG Alexa Fluor 488, ThermoFisher A-21441, 1:500; Goat anti-Guinea Pig IgG Alexa Fluor 647, ThermoFisher A-21450, 1:1000) and DAPI (Thermo fisher) were diluted in 1% goat serum and added to the cells. Cells were incubated for 1 h at room temperature. Afterwards cells were washed 10 times for 1 min with PBS. Cells were imaged using a Zeiss LSM 510 confocal microscope at 10X magnification.

Autaptic hippocampal neurons were fixed at DIV14 in 2% paraformaldehyde (PFA) in culture medium for 10 min and subsequently in 4% PFA for 10 min. Cells were then washed with phosphate-buffered saline (PBS), permeabilized by 0.5% Triton X-100 for 5 min, and blocked with 4% normal goat serum in 0.1% Triton X-100 (blocking solution) for 30 min. Cells were incubated with primary antibodies diluted in blocking solution (anti-MAP2, 1:500, chicken, ab5392, Abcam; and anti-vGlut1 1:1000, guinea pig, AB5905, Merck Millipore) for 2 hr at room temperature (RT). After washing with PBS, the cells incubated with secondary antibodies in blocking solution for 1 hr at RT in the dark (anti-chicken Alexa 568, 1:1000, A11041, Thermo Fisher Scientific; and anti-guinea pig Alexa 647, 1:1000, A-21450, Thermo Fisher Scientific) and washed again. Coverslips were mounted with FluorSave and imaged on Zeiss CellObserver spinning disc confocal microscope (×40 water immersion objective; NA 1.2) with Zeiss Zen Blue 2012 software. Images were acquired as Z-stack and 9 images per plane to capture the whole island in the field of view. The images were post-processed with Zeiss Zen Black software and neuronal morphology was analyzed using SynD automated image analysis (Schmitz et al., 2011 (link)).

The following antibodies and dyes were applied for immunofluorescence (IF) and western blot (WB) analysis: NPHS1 (GP-N2, PROGEN, Heidelberg, Germany, IF 1:300), EPB41L5 (HPA037564, Atlas Antibody, Bromma, Sweden, IF 1:200), YAP1 (14074, Cell Signaling, Danvers, MA, USA, IF 1:200, WB 1:1000), YAP/TAZ (8418, Cell Signaling, IF 1:200, WB 1:1000), ARHGAP29 (sc-365554, Santa Cruz, Dallas, TX, USA, IF 1:50, WB 1:500), EPB41L5 (HPA037563, Atlas Antibody, Bromma, Sweden, WB 1:1000), TUBA (T9026, Merck, Darmstadt, Germany, WB 1:3000), RhoA (2117, Cell Signaling, WB 1:1000), PXN (610051, BD Biosciences, Franklin Lakes, NJ, USA, IF 1:300), Luciferase (NB100-1677, Novus Biological, Bio-Techne GmbH, Wiesbaden, Germany, WB 1:1000), Hoechst 33342, trihydrochloride, trihydrate (H3570, Thermo Fisher Scientific, Waltham, MA, USA, IF 1:1000) Alexa Fluor phalloidin 488 and 555 (A12379, A30106, Thermo Fisher Scientific, IF 1:500). Wheat Germ Agglutinin (WGA)-FITC (FL-1021-5, Vector Laboratories, Newark, CA, USA 1:1000). For immunofluorescence fluorophore conjugated secondary antibodies (A-31572, A-21127, A31570, and A-21450, Thermo Fisher Scientific, IF 1:500) and for western blot HRP-linked, anti-mouse (P0447, Dako, Agilent, Sanata Clara, CA, USA, WB 1:10,000), anti-goat (P0449, Dako, WB 1:5000), or anti-rabbit (7074, Cell Signaling, WB 1:1000) antibodies were applied.