Alexa 555 anti rabbit igg

NSCs were induced to differentiate into neuronal cells for 14 days on 4-well chamber slides (Thermo Scientific Nunc) coated with Matrigel in laminin solution. Cells were fixed with 4% paraformaldehyde (Wako Pure Chemical Industries) for 20 min at room temperature. After washing with phosphate-buffered saline (PBS), the cells were permeabilized by adding 0.1% Triton X-100 and incubated for 15 min. Primary antibodies were diluted to 1 µg/mL for anti-βIII-tubulin (Promega, G7121) and anti-PSD-95 (0.5 µg/mL, Invitrogen, 51–6900) in PBS containing 5% donkey serum and incubated for 1.5 h. After washing, the cells were incubated with secondary antibodies: Alexa 488 anti-mouse IgG (2 µg/mL, Thermo Fisher Scientific) and Alexa 555 anti-rabbit IgG (2 µg/mL, Thermo Fisher Scientific) for 1.0 h. Cell nuclei were stained with DAPI (300 nM). Photographs were obtained using an EVOS FL fluorescence microscope (Thermo Fisher). PSD-95 localization on neurons was imaged on a BZ-X700 (KEYENCE) using Z-stacking to image the steric neuron via BZ-X700 Analyzer software (KEYENCE JAPAN). The images were acquired with a 100 × magnification (Nikon; NA, 1.45) fluorescence objective lens with 8–15 z steps per stack at a step interval of 0.4 µm.

After exsanguination, the mice were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer, and the pancreas, thymus and bone were collected. Rabbit anti-insulin (Cell Signaling Technologies, Danvers, MA USA), rabbit anti-glucagon (Cell Signaling Technologies) and rabbit anti-TNF-α (Abcam, Cambridge, UK) were used as primary antibodies, followed by ImmPRESS reagent anti-rabbit IgG as the secondary antibody. The color was developed using an ImmPACT DAB kit (Vector Laboratories, Burlingame, CA, USA). For immunofluorescence analysis, the sections were incubated with anti-Vcam-1 (Cell Signaling Technologies) or rabbit anti-NG2 (Proteintech, USA) and sheep anti-von Willebrand Factor (Abcam) primary antibodies at 4 °C overnight, followed by Alexa555 anti-rabbit IgG as a secondary antibody (Thermo Fisher Scientific Inc. Waltham, MA, USA). To examine the localization of CD8a T cells in the thymus, frozen sections of fixed thymus were stained with rat anti-CD8a (Clone 53-6.7, Biolegend) as primary antibody and Alexa488 anti-rat IgG (Thermo Fisher Scientific) as secondary antibody at 4 °C overnight. These fluorescent immunostained sections were mounted with a DAPI-containing medium (Vector Laboratories) and observed under a fluorescence microscope.