Alexa fluor 488 goat

5 × 10⁴ cells were seeded into slides (Millipore, MA, USA) and fixed with 4% paraformaldehyde (PFA) for 30 min. Slides were rinsed with PBS for 3 times, blocking slides with 5% BSA for 1 h at room temperature and incubating slides with primary antibodies at 4 °C overnight. Next day, rinsing slides with PBS for 3 times and incubating slides with secondary antibodies in the dark at room temperature for 1 h. Antibodies included anti-ROCK1, anti-E-cad and anti-Vimentin, Alexa Fluor® 488 goat and Alexa Fluor® 555 which were purchased from Abcam, Cambridge, MA, USA were used to IF staining. Visualizing nuclei with DAPI in the dark for 5 min. Analyzing slides by fluorescent microscopy (10x).

Cells in petri dish and microfluidic device were fixed with 2% paraformaldehyde in culture medium for 15 min in the incubator then, replaced with 4% paraformaldehyde in PBS to preserve neuronal structures. They were then treated with 0.1% Triton X 100 for 10 min, and stained with rhodamine phalloidin and actin-stain 488 phalloidin (Cytoskeleton, Inc., Denver, CO, USA) and DAPI for 20 min at room temperature. For immunostaining of Tuj1, VE-cadherin, HB9 and islet1, cells were incubated with rabbit anti-human VE-cadherin (1:200, Biolegend), mouse anti-neuron-specific βIII tubulin (Tuj1 1:100, Abcam), rabbit anti-human HB9 (1:200, Abcam) and rabbit anti-human islet1 (1:100, Abcam) for 1 h at room temperature, incubated with secondary antibodies of Alexa Fluor 555 anti-rabbit IgG (H + L) (1:500), Alexa Fluor 405 anti-rabbit IgG(H + L) (1:500), Alexa Fluor 488 goat anti-mouse IgG (H + L) (1:500), Alexa Fluor 488 goat anti-rabbit IgG (H + L) (1:500) for 1 h at room temperature, followed by three washes in PBS. The neurite elongation length and vasculogenesis of ECs were analysed from the reconstructed three-dimensional images with image analysis software (IMARIS, Bitplane, Switzerland). Cells were observed using a fluorescence microscope (Axiovert 200, Zeiss, Germany) and a confocal laser scanning microscope (FV-1000, Olympus, Japan).

Rats were transcardially perfused with 0.9% NaCl followed by 4% paraformaldehyde in 0.1 M PB (pH 7.4; 4 °C) after deep anesthesia with an intraperitoneal injection of 2% pentobarbital sodium (0.3 ml/100 g). The brains were immediately removed and postfixed with 4% paraformaldehyde overnight. The tissues were then dehydrated in 15% and 30% sucrose in 0.1 M PBS at 4 °C for 48 h. OCT-embedded blocks were sectioned to a thickness of 40 μm. Sections from each group were rinsed in 0.01 M PBS three times and blocked for 2 h with blocking liquid (5% goat serum and 0.2% Triton-100 in 0.01 M PBS) at room temperature. The sections were then incubated with rabbit anti-c-Fos antibody (1:1000, mAb#2250, CST) in blocking buffer overnight at 4 °C. The following day, the free-floating sections were washed with 0.01 M PBS three times and incubated with the secondary antibody Alexa Fluor 488 goat anti-rabbit (1:500; ab150077, Abcam) at room temperature for 2 h. DAPI was applied for nucleus staining for 5 min. After being washed in 0.01 M PBS, the brain slices were cover-slipped. Images were captured using a microscope (Axio Imager.A2, ZEISS, Germany), and analysis was performed using NIH Image J software (Bethesda, MD, USA).