E15.5 mouse neurons were transfected using Lipofectamine 2000 2 hours after plating, with 220 ng of Pbird GFP with 500 ng of mycEV, mycRBBP7WT siRNA-resistant, or mycRBBP7C166S siRNA-resistant plasmids. Cells were cotransfected with 400 nM of each siRNA. Media were changed 3 hours after transfection to NB media supplemented with 0.33% B27, 1 mM glutamine, 1× penicillin-streptomycin, and 10 μM FdU, and coverslips were fixed in 4% paraformaldehyde/PBS 48 hours after transfection. Coverslips were washed once in PBS, permeabilized in 0.3% Triton X-100/PBS, blocked in a 5% normal goat serum/5% FBS/PBS solution for 1 hour at RT, and incubated with chicken anti-GFP (ab13970, Abcam) overnight. After three PBS washes, samples were incubated with 4′,6-diamidino-2-phenylindole and Alexa Fluor goat anti-chicken 488 (A11039, Life Technologies) at 1:1000 for 1 hour at RT. Coverslips were washed and mounted. Slides were then blinded before imaging and analysis. Images were acquired using an SPE or SPE3 confocal microscope (Leica) with LAS AF software and processed using ImageJ/Fiji. Sholl analysis was performed using ImageJ Simple Neurite Tracer (fig. S8) or a Fiji Sholl Analysis plugin (Fig. 4), and samples were deblinded after final processing.

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



Q&A
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.