I have fixed brain tissue that I have cryopreserved using a sucrose gradient, but at this point I would like to go in a different direction...I plan to use a vibratome to section the tissue and then stain it (IHC). I can't think of any reason this would be an issue, but I wanted to ask the experts.
I can't think of any reason the vibratome wouldn't work with that tissue. It should be fine, I hope.
you kinda do not have a choice with brain tissue. I would give it a try, from my experience - it should work. It should not interfere. Good luck!
Don't worry. Sucrose is essential for cryocutting but it will not interfere with tissue quality if you decide to use vibratome.
I have done this many times with mouse brains that were fixed and then sunk in 30% sucrose. I had great results. It actually helps as it firms up the tissue for cutting.
You should wash four times your samples in 0.01 M PBS buffer.
good luck, Ania
Sucrose isn't fixative of the sample but it conserves the structure of the cryotissue and non cryotissue, for a little while of the time at 4°C.
It is a cryoprotectiv for membrane cellullar to create a disidratation of the cell, so the water leaves and minimized te crystallization.
It can be used at 2% with 4% PFA in phosphate buffer solution.
Surely you can go in this direction using the vibratome, but remember to wash the sample in PBS and fix it
Any tissue after cryopreservation can be washed in PBS pH 7.4 and fixed.
If you need to perform an IHC, I would recommend to change the method, which would use a classical fixation in Zinc-formalin or Formol-buffered saline at pH 7.4 and then after the dehydration, the sample would paraffin-embedded tissue for microtome cut.
Obviously depends on the IHC method that is whether the antibodies are also tested for paraffin-embedded tissue.
As the others have already suggested, you should be able to get good sections. But depending on the direction that you are taking this project, I've certainly had some antibodies that prefer one method or the other (EITHER sucrose, OR perfusion).
Hopefully the antibodies that you are using "like" this new treatment and work as expected. Certainly, it will become difficult to compare any counts from this method with counts derived from non-sucrose cryoprotected tissues....
Thanks, all! As a follow up, any tips on whether or not you can section fixed, paraffin-embedded tissue on a vibratome? I am guessing not...just trying to get the most our of my tissue. Thanks!