I'm trying to image the ventral nerve cord (VNC) and some neuronal projections labeled with mVenus. Although I can see very well the endogenous labelling of mVenus, I’m having a lot of problems with the monoclonal mouse nc82 (anti-BRP) labelling. This epitope (BRP) is widespread so I should see it strongly all over the place. As you can see below the labelling is very heterogeneous with some regions without signal.
I tried already several things like increasing the incubation time with the antibody for 3 days and increasing its concentration, but it did not improve greatly. I also tested the antibodies for their quality.
Did someone else have this problem? Do you have any ideas why I cannot have a good labelling?
Very briefly this is my protocol:
After a brief fixation with PFA4%, I dissect the VNCs in PBT0.03% and put them directly in 4% PFA for a fixation of at least 30 min and then rinse with PBT 0.5% 5x 20min. After 30 min blocking (5%NGS) I incubate with the primary antibody (1:10) in blocking solution for 3 days at 4ºC. After washing with PBT 0.5% I incubate another 3 days with secondary 1:500. In the end I rinse again with PBT and mount in Glycerol.
Thank you for your help!
This is an interesting result and it is indeed strange that it is so scattered. Are you doing all of these steps on a rocker or shaking platform?
This was my approach (Doll et al 2017): Brains were dissected in 1xPBS, fixed for 30 minutes in 4%PFA/ 4%sucrose, washed 3×20 minutes with 1xPBS, and blocked for 1 hour in 1xPBS/1%BSA/ 0.5%NGS/0.2%Triton-X 100. Brains were then placed in primary antibody on a shaking platform at 4°C overnight. mouse anti-Bruchpilot (nc82, 1:100; Developmental Studies Hybridoma Bank (DSHB)). Following 3×20 minutes washes in 0.2% PBS-Triton, brains were incubated in secondary antibody for 2–3 hours. Secondary antibodies: anti-mouse-IgG AlexaFluor 568 (Molecular Probes, Eugene, OR), all used at a 1:500. Following 3×20 minute washes in 0.2%PBS-Triton, 1×20 minutes in 1xPBS and 1×20 minutes in dH20, brains were mounted in Fluoromount G (Electron Microscopy Sciences, Hatfield, PA).
Your incubations are very long, to my eye. Again the first thing that popped in my head was the shaking platform ... I hope this helps.
Yeah I was doing an overnight incubation but apparently all my colleagues do 3 days with this ab. Yes I use a shaker. Thank you for the protocol, I will try.
Hi Alexandra M. Medeiros,
It might be a bit late for answering your question, but it happened to me once with Brp and I solved it by changing the secondary antibody. I was initially using the Alexa 5XX and when I changed it to the Alexa 488 it worked like a charm.
I hope it helps you,
All the best
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