I want to measure or identify the level of oxidative stress in different cell types of mice testis upon inducer. I want to do so in two ways:
1) Testicular tissue will be cultured in an organ culture system in the presence of inducer,
2) Injection of inducer in in-vivo condition, followed by isolation of testis.
For both the cases testicular tissue and testis will be fixed in formalin to make a tissue section on a slide.
Does anyone have any idea how to look for oxidative stress in the above conditions?
You can use immunohistochemistry for this. There are various antibodies for detection of markers for oxidative stress end products of lipids, proteins and DNA. 4-HNE, nitrotyrosine, 8-OxoG and so on.
Try to estimate protein carbonyl content, its a very stable molecules. Amount of protein carbonyl represents the extent of oxidative stress.
Dear Rajesh, Thanks for your suggestion, but I want to look for specific oxidative stress by IHC technique, in individual cell type of testis ( which contains different type of cell type) in tissue section. Could you please send any reference which suits my need.Thanks in advance
immunohistochemical analysis is best to detect oxidative stress in parafinized section , we can try protein expression of HSP70 . that also shows tissue respond to stress
Please keep in mind that you can not detect the ROS levels directly in formalin fixed tissue, since the life-time of ROS is very short (less than 1 ms). A lot of methods mentioned above have significant limitations and may provide you wrong informations. You can detect the product of ROS which is relatively stable in the fixed tissues.
You can measure ROS, e.g using DHE, in frozen tissue sections or cell pellets embedded in OCT.
I need to measure the most constant molecule of ROS in salivary glands tissue of pleomorphic adenoma fixed in formalin.
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