Our lab has recently gotten access to the Muromachi Microwave Fixation system which kills and stops all enzymatic activity within 1 second! VERY neat equipment, however, I am having difficulty finding a published protocol on how to prep my brains for sectioning and immunofluorescence.
Normally, we perfuse our animals which clears all the blood from the vasculature (this is important to avoid autofluorescence during IHC/imaging). Then the dissected brains go into 12% sucrose in PFA for a day, get frozen, then they are ready for sectioning and IHC-F.
With microwave fixation, the blood is not cleared from the vasculature. I'm wondering if anyone has used this technique for IHC-F before, and whether you would be willing to share your protocol!
Unfortunately, I don't think that the intended use of the Microwave Fixation Machine and your end points are compatible. Certain elements of the process may need to be altered, potentially profuse/ flush blood and then attempt to "fix" the tissue. However I'm not sure that the tissue is truly fixed and that only the enzymes are deactivated, and therefore many downstream processes may be adversely effected. I'm guessing by the relatively new technology and nuanced niche this machine is marketed to precludes using it in the fashion you are suggesting. I hope you can derive a protocol to further develop this technology, and good luck!
Thank you for your response, Jeffrey P Cheng
Maybe you're right, these two are not compatible. We already tried treating the tissue sections with various protocols using hydrogen peroxide to eliminate the peroxidase enzymes found on blood cells that cause the autofluorescence. They didn't work.
Maybe one day we'll figure it out!
I agree with Jeffrey. I would not use microwave for fixation. If you are flushing the vessels of your experimental animals I would recommand to perfuse them with PFA and cryoprotect them afterwards with 30 % sucrose. The most intensiv autofluoresce you will have in the green channel ( 488 nm). Depending what you like to investigate I would recommand to use red Channels like 546nm ( Cy3) or dark red like 647nm (Cy5), 750 nm Cy7. In all this cases you will have less autofluorescence.
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