Hi Everyone,
Have you ever experienced a massive amount of small holes on an FFPE mouse brain section (as showed in the attached)? and do you know what could cause this effect?
The mice were normal WT C57BL. The brains had been perfused with PFA and then fixed for 24 hours in formalin. They were further stored in 70% EtOH at 4C for a few weeks before embedding. The cutting thickness was 5 µm.
I observed these sponge-like tissue sections in a few samples. However, other samples were completely alright.
Thank you very much!
Dear Chen!
Such small holes and voids can be the result of a thin sagittal cut, when the cells in the tissue do not adhere tightly to each other (the result of cell damage, dead destroyed cells, cavities filled with fluid upon fixation, when the tissue contains a large amount of fluid - the brain).
Also these holes may often result from following reasons. (1) using unhealthy or necrotic tissue; (2) deficiencies in fixation; (3) using damaged blades or knives
It is problem with fixation, may be due to too think tissue samples, or bad fixation solution.
Thank you very much for the answers Alexandr Chernov and Dmitry Zinovkin. So the issue could be under fixation? We ruled out the possibility of damaged blades, as the bad tissues had these small holes all through the brain, and the good tissues had nice morphology all through.
Dear, @Chen Zhang, blades cause mostly line scratches. It looks at higher magnification like pericellular oedema of the brain. https://www.researchgate.net/publication/11488446_Spontaneous_mycotoxic_nephropathy_in_Bulgarian_chickens_with_inclarified_mycotoxin_etiology/figures?lo=1
https://www.researchgate.net/publication/264629102_The_Potential_Role_of_Morus_Alba_Leaves_Extract_on_the_Brain_of_Mice_Infected_with_Schistosoma_Mansoni/figures?lo=1
Thank you for the follow-up Dmitry Zinovkin. Yes, the morphology of the bad tissue indeed resembles the pericellular edema of the brain! That could potentially be caused by the PFA perfusion at the termination (i.e. too high perfusion pressure)?
Dear Chen Zhang, I agree with you and suppose that high pressure during perfusion could be a possible cause of this artefact.
Dear Chen Zhang,
Have you solved this problem? I have the same issue when I fix and do the brain section. Could you please share your protocol with me if you have found the solution?
Thank you very much
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