I want to perform IHC assay on tissues extracted from mice. My question: Is it better to perfuse the mouse first before extracting the tissues? or to extract the tissues first then fix them immediately in PFA? Which technique is better? And is there a big difference between both techniques regarding the sensitivity of the test? Giving that I am interesting in detecting the up and down release of certain proteins, and any small difference would influence the results interpretation.
Thanks in advance!
I would certainly do a intracardiac perfusion first and than harvest the tissues. Otherwise it takes a longer time for fixative to penetrate deep into the tissues and stabilize the proteins of interest. Better fixed tissue will be easier and better to make sections from. Another factor is that you will be removing all the blood from the tissues and that will reduce your background.
best wishes, Refik
Alaa, what is your tissue type ? I suggest perfusion and postfix with the same solution-paraformaldehyde in 0.1MPBS
Hi all, thank you very much for your precious replies! Much appreciated.
I want to collect Skin, Spinal Cord and DRGs samples from which we need to make sure that the levels of the proteins in there are not changed to detect them with IHC. What about PCR WB and ELISA? Do I need to perfuse as well? Thanks again!
Hi Alaa,
For PCR, WB and Elisa, you can just collect fresh tissues or freeze them immediately.
good luck, Refik
Hi. If you want to use the same set of animals for procedures such as PCR and histological procedures such as H&E, Immunohistochemistry etc. you should not perfuse, but collect fresh tissues freeze immediately for PCR and immerse other samples immediately in the fixative you have selected to be use 4%PFH. Alternatively and depending of the aims of your investigation have more animals following the same protocol one 1/2 of the animals to be used for tests where tissues should be collected frozen and the other 1/2 perfuse and tissues are for exclusive use of histopathology. The cost of having two set of animals is usually a problem. We have done by setting collaboration with other investigators and sharing the cost. Good planing ahead of time avoids a lot of headaches. Do not allow tissues to authorize for any reason as the will be damaged.
I enclose one of our publications with more details of the methodology used.
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