I'm trying to work through the protocols for TUNEL staining using In situ Cell Death Detection Kit (Roche), and the recipe of the permeabilization solution keeps bugging me. I admit, we never used sodium citrate in other assays before, especially that of immunohistochemistry, tissue nor cell. We simply added Triton X-100 to PBS in both of these cases. I searched the web and I found a lot of recipes included these particular compounds, but nobody explained why it's there or what it's for. Can anybody shed some light on this matter?
Hi Yun Li
I know that Citrate Buffer is an Antigen Retrieval Protocol and is commonly used when there are Formalin or other aldehyde fixation forms in animals.
The citrate based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies.
Hope the answer can help you.
Hello Yun Li, Juan Munoz is absolutely right. Step of citrate buffer incubation will enhance your staining intensity. Also, I would like to mention here that incubation of cells/ tissue in citrate buffer should be on high temperature (ranges-90-110 degree centigrade). Thus due to boiling your cells/tissue may damage somewhat, But this is limitation of this step. Also, you can make a plan to adjust your cells/tissue in such a manner so that less damage occurs. I have some experience about that, during my master study , I used to fix tissue section slide in fixed slot and then used it for boiling in a large beaker. This citrate buffer boiling step is only needed for formaline/other fixed cells/tissue, however if you have cryopreserved cells/tissue then you can escape this step.
Good luck!
On top of sodium citrate for AR protocol, you can also use pepsin digestion, milk powder or even alcohol. But you have to remember that alcohol is also a fixative and may form new crosslinks
Hey Yun Li, I know this was a while ago, but I am using the TUNEL on cryopreserved sections as well and was having some trouble with the sodium citrate. So, did you skip that step, or use something else in its place?
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