High and low autophagic fluxes will be induced in mouse brain using Intraperitoneal injections. What is the best method of fixation to use in order to dissect brain areas of interest afterwards? What is the best method of sacrifice to preserve tissue of interest. Should sacrifice commence before or after fixation?
I used to perfuse animals with fixative after flushing out the blood with buffer, using a syringe or peristaltic pump. You would need to be authorised to do this because the animal is anaesthetised and the heart should be beating. A needle is inserted into the left ventricle of the heart and the right atrium is punctured to release the blood and fluids. In the UK you need a personal animal licence. The mice need to be anaesthetised at the start using pentobarbitone (that's what I did).
This method preserves the brain very well, and I used it for pituitary as well, in fact many tissue benefit from perfusion fixation.
I agree with Jennifer that the perfusion is the best way for brain fixation. The thing I would add is, that you might want to flush the blood with 10 Units/ml of cold Heparin, which will reduce the clotting and then switch to fixative.
If you are using formaldehyde as a fixative you will need to postfix the brain after it is isolated. This fixative is penetrating at the rate of 3.6 times the square root of time so it takes a while to reach the whole brain even by perfusion. The crosslinking of proteins is another thing and it takes even longer (hours to days). Therefore the standard procedure for brain preservation is 30min-1h perfusion with 4% formaldehyde solution, isolation of the brain and than overnight-24h postfixation. Bear in mind that after 24h fixation formaldehyde can be still washed out from the tissue (Helander, 1994), so it is good to avoid long storage of the tissue in PBS after it has been fixed. Cut it or embed within a day.
Also it was postulated, that perfusion may lead to some shrinkage and distortion of brain structure, because of the influx of sodium ions and consecutive swelling of the cells. To reduce that it is suggested to use 10% sucrose solution at 300 mm Hg pressure before perfusing with fixative (Cragg, 1980).
I hope this will help you a bit. Good luck.
Thank you for all the tips!
Also, is there any alternative to fixing? I would also like to assess a dynamic process of which the rate cannot be quantified once the brain is in a fixed state, so ideally I would like to preserve the brain, dissect brain areas of interest and treat the brain tissue with specific inhibitors and inducers. How reliable would liquid nitrogen freezing be for this purpose? Have you guys done anything like this before?
As far as I know snap frozen tissues retain a lot of their enzymatic activity. Do you want to culture the tissue after the dissection or you are planning some biochemical assay? If the former, you can directly dissect the unfixed brain and transfer the regions of interest into coulture dish. Otherwise you can also try vibratome sections. As long as the tissue is immersed in culture medium and you provide aeration you are fine. For the freezing technique I'd suggest to drop your tissue into isopentane chilled in liquid nitrogen. It gives you better thermal conductivity than liquid nitrogen itself and hence less chance of ice crystals formation.
Great that helps a lot! Yes ideally I would like to culture the tissue, was afraid the brain will start disintegrating too fast to dissect and culture, but will definitely try your suggestions! Thank you so much Agnieszka!
If you do the dissection on ice you should have easily 30 minutes before you get some real damage. For longer manipulation you'd need to think of supplying nutrients and oxygen. At least for DRG culture the tissue can be dissected into cold PBS and then transfered to the proper culture medium and put into the incubator.
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