Need help on principles

Claire_Zhang

The hydrogel is the framework of the CLARITY sample, and it can fix almost all the protein in situ even under the ETC electric power. But how could the labeled antibody get across the network towards the target molecules? And how could them be removed thoroughly? Thank you!

Andy_Donaldson

Once the lipid has been removed from the tissue sample (via the ETC step), all you have left is the polymer meshwork that holds the biomolecules of your tissue in place (morphology is preserved very well). This meshwork is actually more "open" than uncleared tissue. As a result, antibodies move readily through the meshwork deep into the tissue. Bear in mind though, that even though antibodies move easily through the meshwork, a thick sample will still take time. Check out the section of the literature entitled "Immunostaining of CLARITY-processed mouse brain tissue." This section gives a good idea of time frame for immunohistochemical staining.

Fabio

Also you wash your samples with 4% SDS this is enough to wash out any bound antibody.
This way you can stain multiple times.