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4 protocols using alexa flour 594 donkey anti rabbit igg

1

Immunofluorescence Staining of MCF-7 Cells

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MCF-7 cells were cultured on cover slips. Following washing twice with PBS, cells were fixed with 4% paraformaldehyde for 15 min. Cover slips were washed three times with PBS and the cells were permeabilized with 0.1% Tween-20 in PBS for 20 min followed by blocking for 30 min using blocking buffer (5% bovine serum albumin in PBS). Following overnight incubation with the primary antibodies, the cover slips were washed three times with PBS and treated with Alexa Flour 594 donkey anti-rabbit IgG (A21207; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h in the dark. Cover slips were then washed three times in PBS and mounted with VECTASHIELD Mounting Medium with DAPI (H-1200; Vector Laboratories, Burlingame, CA, USA). Images were captured using a Leica confocal laser scanning microscopy system (Leica Microsystems GmbH, Wetzlar, Germany).
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2

Immunohistochemical Analysis of PAC1R

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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).
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3

Biotinylated Dextran Amine Axon Labeling

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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).
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4

Immunostaining of MeCP2 and β-Endorphin in Brain

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Immunostaining for MeCP2 and β-endorphin were performed as described previously [17] (link). Brain sections (20 µm thickness) were cut on a cryostat (Leica) and collected on pre-chilled glass slides. These sections were double stained for MeCP2 (1∶500) and β-endorphin (1∶200) antibodies. The primary antibodies used were mouse anti MeCP2 (Abcam, Cambridge, MA) and rabbit anti β-endorphin (Bachem, Sam Carlos, CA). Secondary antibodies used in this study were Alexa-Flour 488 donkey anti-mouse (2 mg/ml; Invitrogen, Grand Island, NY) and Alexa Flour 594 donkey anti-rabbit IgG (2 mg/ml; Invitrogen, Grand Island, NY). After staining, pictures were taken using a confocal microscope with a 20× objective (Nikon EZ-C1 3.60 build 770, Gold version; Melville, NY). Total number of β-endorphin cells as well as total number of β-endorphin cells, located on the right and left side of the third ventricle, that were positive for MeCP2 were counted. The experimenters were blind to the experimental treatment group of the section during counting.
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