I already tried to use increasing amounts of tween or triton x and have tried waiting 3 nights. My other antibodies penetrate through the tissue just fine, BRDU and NeuN.
Thank you in advance!
The first image is a gallery of the z stack was using 1 hour blocking and .1% tween. The stain fades in the middle of the slice.
The second image is using .2% tween
the third image is .3% tween
Hi Tatyana,
how thick are your sections? You mentioned you have tried to incubate over 3 nights. With both first and secondary antibodies, or only the first? If you are interested to label the whole thick section of around 100-200 µm my tip is the following: start from using 1%Tx (w/woNGS or BSA) and block for 2-3 hours, incubate with your first AB in the 0.3% Tx for up to 5-6 days (add 0,1% Sodium azide to prevent the fungi), wash and then incubate with the secondary for up to 2 days. Good luck! Natalia
Hi Tatyana, we regularly use 1% DMSO when staining floating slices (from 20 um to 40 um). When we have really thick sections or whole pieces of tissue (for spim or light sheet microscopy) we go up to 2-3% DMSO to increase the penetration. The main problem in slices are not the membranes, which you take care of with triton (always keep it, in blocking, antibody dilutions AND washes, because the micelles are trapped in the tissue and can always reclose), but the proteic extracellular matrix, on which triton has no effect, but DMSO can take care of. We use it during blocking, but also in the antibody dilution, both primary and secondary. Give it a try... and good luck!
.3% is not that high. I use for 1% for two hours at least, and make sure to keep at least 0.1% Triton in all subsequent solutions to decrease surface tension and increase penetration. What solution are your primaries in? Dcx is a tricky one at the best one times anyway. Agree with Natalia further up is that doesn't do the trick..
- Badges
- Science topic
- Similar topics
- Agriculture
- Horticulture
- Seeds