Also in the Article

For immunofluorescence stainings against ZsGreen, POMC, tdTomato and EGFP, all incubation steps were performed at room temperature unless otherwise stated.

Floating sections were washed once for 10 min in PBS, incubated for 10 min in 0.3% glycine, washed again for 5 min in PBS and incubated for 10 min in 0.03% SDS/PBS. Subsequently, sections were blocked for 1 h in 3% donkey serum in PBS containing 0.25% Triton X. Next, sections were incubated overnight at 4 °C in primary antibody diluted in Signal Stain (Cell Signaling, 8114). Primary antibodies and dilutions used were: rabbit anti-ZsGreen (Takara Bio Clontech, 632474; 1:100), rabbit anti-POMC (Phoenix, H-029-30; 1:1,000), rat anti-mCherry (for tdTomato; Thermo Fisher Scientific, M11217; 1:1,000) and chicken anti-GFP (Abcam, ab13970; 1:1,000) were used. The following morning, sections were washed three times for 10 min in PBS containing 0.1% Triton X and incubated for 1 h in secondary antibody in PBS containing 0.25% TritonX. Secondary antibodies and dilutions were donkey anti-rabbit Alexa Fluor 488 (Thermo Fisher, A21206; 1:500), donkey anti-rat Alexa Fluor 594 (Jackson ImmunoResearch, 712-585-153; 1:500) or goat anti-rabbit Alexa Fluor 594 (Thermo Fisher Scientific, A11012; 1:500) and goat anti-chicken Alexa Fluor 488 (Thermo Fisher Scientific, A11039; 1:500), for 1 h at room temperature. After three washing steps for 10 min in PBS containing 0.1% Triton X, sections were mounted in Vectashield DAPI-containing mounting medium (Vector Laboratories, VEC-H-1200) and stored at 4 °C in the dark until imaging.

Note: The content above has been extracted from a research article, so it may not display correctly.

Also in the Article

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.