I am performing fibronectin staining on Xenopus embryonic tissue. The tissue is fixed overnight in 4%PFA, permeabilized in 1xPBS with 0.3%TritonX, Blocked in 10%Blocking in 1XPBS with 0.3%TritonX, Fibronectin antibody 4H2-DSHB used at 1:50 dilution, washed in 1XPbs with 0.1%Tween20, secondary antibody in 1xPBS with 0.1%Tween20 overnight at 4*C, washed in 1XPBS with 0.1%Tween20.
The staining is getting restricted to the cells on the periphery of the tissue. The staining is quite specific. However, the cells at the center and away from the periphery are not slowing any signal.
Assumption: Antibody not penitrating inside the tissue.
Any advice on how to solve this problem?
Hi Aritra,
your protocol generally sounds good.
Some quick questions, maybe helping to identify next steps:
Did you test your antibody in this dilution also on thin sections to check whether the antibody itself really works?
How long is your permeabilization step and incubation with first antibody?
In what color is your secondary? Is it fluorochrome-labelled (Which?)?
Did you try clearing of tissues?
I am just asking, because for example....
For my whole tissue stainings, I usually stain and wash very long! Over night or even over the weekend in buffer containing tween or triton.
I permeabilize using the eBioscience FoxP3 Fix/Perm kit, but tween/triton should work fair enough.
Be aware that if you do fluorescence microscopy, short wavelength emitters are more absorbed unspecifically. In contrast long emitters can give you better signals in deeper layers. Meaning for example AF647 and/or AF594 are the better choices over FITC/AF488/pacific blue etc. Of course this depends on your microscope
We often clear tissue. I for example use iohexol (commercial name "Omnipaque") to clear samples over night and would also mount samples afterwards in this. Quite cheap stuff. It is also the basic component of a published clearing protocol called "SeeDB2".
Best wishes,
Urs
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