Alexa flour 594 donkey anti rabbit igg

The cryosections were washed with phosphate-buffered saline (PBS) and then blocked with 5% horse serum in PBS for 1 h and incubated overnight at 4 °C in a solution of the following primary antibody: rabbit anti-PAC1R antibody (1:200, developed by our laboratory) [55 (link)]. It was then washed with PBS and reacted with a secondary antibody: Alexa Flour 594 Donkey anti-rabbit IgG (1:800, Invitrogen, MA, USA) for 60 min at room temperature. It was re-washed with PBS, followed by nuclear staining with DAPI (1:10,000, Invitrogen) for 3 min at room temperature; then, it was washed with PBS and encapsulated using a VECTASHIELD Vibrance Antifade Mounting Medium (Vector Laboratories, Newark, CA, USA). After drying, the samples were visualized and imaged on a fluorescent microscope (BZ-X710; Keyence, Osaka, Japan).

For biotinylated dextran amine (BDA) immunohistochemical analysis of axon labeling, we embedded the C1–C4 spinal cord regions in OCT as described above and prepared 30 μm sections. These were immersed in 0.3% TritonX-100 for 30 min, then incubated with streptavidin coupled to Alexa Fluor 488 (1 : 500, S11223, Invitrogen) at room temperature for 1 hour. For BDA/p-S6; BDA/SYN double-labeled immunofluorescence staining, we sectioned in the same manner. The primary antibody was rabbit anti-p-S6 (1 : 200, Cell Signaling Technology) and mouse anti-SYN (1 : 200, Cell Signaling Technology). The secondary antibody was Alexa Fluor 488 streptavidin (1 : 500), Alexa Flour 594 donkey-anti-rabbit IgG (1 : 500, A21207, Invitrogen), Alexa Fluor 488 streptavidin, and Alexa Fluor 594 goat-anti-mouse (1 : 500, Invitrogen). Fluorescence was detected using a fluorescent microscope and a two-photon laser scanning microscope (LaVision BioTec, TriM Scope II, Germany). We selected and analyzed three nonoverlapping visual fields from each section. The results showed optical density (OD).