After the fixation with 4% paraformaldehyde (PFA), I assume that everything that is fixable by PFA will be preserved while those did not will lost much of the attention from the investigator (may due to degradation or assuming lost of physiological characters).
Interestingly, according to what I read/experienced so far, people usually use saline or its associated form as the main buffer solution during the histological staining procedure, particularly for the immunostaining parts.
I was wondering what would be the main purpose/advantage of using saline at the post-fix stage. Additionally, I was wondering whether people had tried using ddH2O instead of saline in the steps which originally would use saline.
Thank you very much in advance!
The immunistaining (as the name suggests) is performed with antibodies which interact noncovalently with the binding epitopes. Thus in order to stabilize the antibody and the binding epitope and to make sure that the non covalent interaction takes place as desired, physiological ion concentrations are necessary. Furthermore in ddH2O proteins (like antibodies) would unspecific adhere to all kinds of structures due to electrostatic effects, which are shielded by the free ions in the saline.
The immunistaining (as the name suggests) is performed with antibodies which interact noncovalently with the binding epitopes. Thus in order to stabilize the antibody and the binding epitope and to make sure that the non covalent interaction takes place as desired, physiological ion concentrations are necessary. Furthermore in ddH2O proteins (like antibodies) would unspecific adhere to all kinds of structures due to electrostatic effects, which are shielded by the free ions in the saline.
Dear Ping:
You need a buffer to avoid pH changes in your sample, you need salts to avoid electrostatic interactions, and you need a buffered and ion-containing medium (that must be at the physiological concentrations at which antibodies work) for your antibodies and their antigens in the tissue to be stable and able to interact with one another.
I agree with Francisco - we need ion-containing buffer solution for 1.- to keep osmolarity, 2.- to keep pH level and 3.- antibodies need of ion-contained buffer to interact with antigens
For an additional reason to the ones mentioned above, you need to wash off the excess solution you add to your sample in each step; an appropriate buffer can do that job.
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