quality of fixed frozen sections - why do they turn black? (Jul/11/2007 )
I am attempting to section rat brain tissue for subseqent laser capture microdissection. I fix the tissue in 4% PFA, freeze in OCT, then section using a cryostat into 30 micron slices. Immediately after getting a slice, I collect on a cold slide and melt it into place, then refreeze it using dry ice. After all my slices are collected onto slides I dry them in a vacuum dessicator for 24 hours.
My problem is that some of the slices look pretty bad under the microscope. They are very dark - some almost all black, and little morphology can be seen. What I can't figure out is why this does not happen with all of the slices. Some of my slices are a nice light clear tan color and all morphology can easily be seen.
The same thing occurs when I have cryoprotected overnight in 30% sucrose. Some are okay, some are not.
Does anyone have any idea on why this blackening could be occurring?
Thanks!
My problem is that some of the slices look pretty bad under the microscope. They are very dark - some almost all black, and little morphology can be seen. What I can't figure out is why this does not happen with all of the slices. Some of my slices are a nice light clear tan color and all morphology can easily be seen.
The same thing occurs when I have cryoprotected overnight in 30% sucrose. Some are okay, some are not.
Does anyone have any idea on why this blackening could be occurring?
Thanks!
hallo Jennybens,
I think you don´t have to treat the tissue with 4% PFA before embedding in OCT. PFA is only used when tissues are embedded in paraffin for crosslinking of proteins (lysin-crosslinking).
We use our OCT cryopreserved tissue samples for IHC and it works very well. How do you freeze the tissue? It should be very gently, therefore usage of 2-Methylbutane is recommended.
Step by step:
1. Place tissue into embedding mold, place it so that you know your first cut
2. Immerse tissue with OCT
3. Place the mold into dish with 2-methylbutane
4. place this dish into liquid nitrogen and look how it freezing (from the out- to the inside, very slowly), store at -20°C until use
5. sections could be made when needed
And we only dry the tissue slides before usage, which means that the slides should be used immediately ore stored at -20°C (we wrap them with aluminium foil).
sorry, but i could not help you with the sucrose method.
I hope, this helped.
How did the tissue block look? Are these in the same color grade, or some are draker than others?
I wonder what will happen if you fix the tissue by perfusion followed by at least 4 hr in 4% PFA, soaked in 30% sucrose o/n before OCT and freeze, sections.
Hi guys. Have either of you ever frozen bones before and cut cross sections from them im having trouble.
thanks