Cardiac fixation used to preserve mouse and rat submandibular gland sections. Would the ATP content be detectable? Aside from variability in section thickness, what should I be wary of?
Because ATP can not be fixed by PFA, it is possible to detect ATP in fixed tissue. The important thing is that how did you preserve your samples? What kind of buffer system did you use? ATP is highly soluble in water and is quite stable in solutions between pH 6.8 and 7.4, but is rapidly hydrolysed at extreme pH. ATP is an unstable molecule in unbuffered water, in which it hydrolyses to ADP and phosphate. This is because the strength of the bonds between the phosphate groups in ATP is less than the strength of the hydrogen bonds (hydration bonds), between its products (ADP + phosphate), and water.
Thank you for your response; this makes absolute sense, chemically. The samples are preserved in the same fixative as was used for the cardiac fixation, which is HEPES-based. So if I put ~150 um sections into a 96-well plate and perform the luciferase assay directly on them, you think it will work?
It maybe OK by using 150um sections, but the signal might be weak. Because the thickness of 150um is composed by several layers of cell, luciferase and drugs may difficult to penetrate into cell. It is better to release substrate from cells by homoginization before luciferase assay. I believe the signal will change more than 10 times before and after cell disruption. And please wash out fixative from tissue precisely before you doing luciferase assay.
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