For double labelling of c-Fos and orexin, tissues were washed and incubated with a PBS cocktail consisting of 2% NDS, 0.3% Triton X-100, rabbit anti-c-Fos (1:100 of Cat#Sc-52, Santa Cruz Biotechnology, Inc. and/or 1:1000 of Cat#ABE457 Millipore Corporation, Temecula, CA) and goat anti-orexin (1:100 of Cat#Sc-8070 Santa Cruz Biotechnology, Inc.) antibodies at 4°C for 48-72 h. The sections were then washed and incubated in Alexa Fluor 594 donkey anti-rabbit secondary antibody (1:100; Jackson ImmunoResearch Laboratories Inc.) in 0.1 M PBS for 2½ h. After washing in PBS, sections were incubated with Cy™ 5 donkey anti-goat secondary antibody (1:100; Jackson ImmunoResearch Laboratories Inc.) for 2½ h. Finally, the sections were rinsed in PBS and cover-slipped using Vecta Shield (Vector Laboratories) anti-fade mounting media.
Cy 5 donkey anti goat secondary antibody
Manufactured by Jackson ImmunoResearch
Cy™ 5 donkey anti-goat secondary antibody is a fluorescently labeled antibody that binds to goat primary antibodies. It is used in immunoassays and imaging techniques to detect and visualize the presence of target antigens.
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2 protocols using cy 5 donkey anti goat secondary antibody
1
Double Immunofluorescence Labeling of c-Fos and Orexin
2
Double Immunofluorescence Labeling of c-Fos and Orexin
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For double labelling of c-Fos and orexin, tissues were washed and incubated with a PBS cocktail consisting of 2% NDS, 0.3% Triton X-100, rabbit anti-c-Fos (1:100 of Cat#Sc-52, Santa Cruz Biotechnology, Inc. and/or 1:1000 of Cat#ABE457 Millipore Corporation, Temecula, CA) and goat anti-orexin (1:100 of Cat#Sc-8070 Santa Cruz Biotechnology, Inc.) antibodies at 4°C for 48-72 h. The sections were then washed and incubated in Alexa Fluor 594 donkey anti-rabbit secondary antibody (1:100; Jackson ImmunoResearch Laboratories Inc.) in 0.1 M PBS for 2½ h. After washing in PBS, sections were incubated with Cy™ 5 donkey anti-goat secondary antibody (1:100; Jackson ImmunoResearch Laboratories Inc.) for 2½ h. Finally, the sections were rinsed in PBS and cover-slipped using Vecta Shield (Vector Laboratories) anti-fade mounting media.
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