How to effectively cryosection organotypic mouse brain slices?
In our lab, I am currently trying to establish a demyelination model using ex vivo brain slices. I am using 6-8-week-old mice to collect their brains and slice them at a 400 µm thickness using a vibratome. I then maintain them in culture for 10 days on a membrane insert (using an air-liquid interface approach). With this protocol, I aim to observe the differences in MBP (myelin basic protein) expression in the corpus callosum over time and at different concentrations of our demyelinating drug.
To do this, I fix the slices with 4% PFA overnight and cryopreserve them in 30% sucrose for two days (until they sink). Afterwards, I collect the slices, place them flat in an OCT mould, and embed them in 100% OCT. However, I noticed that the tissue does not stay flat at the bottom; as a result, I am only obtaining tissue fragments during sectioning.
I saw that some papers use a 50% OCT / 50% sucrose-in-PBS solution for embedding. I also considered using whole-mount staining to avoid sectioning the tissue, but in my case, I need serial sections for different assays.
Does anyone have any experience with this procedure or could help me with this issue? It has been really challenging to overcome this small step, which ends up compromising everything because, ultimately, I cannot actually analyse my tissue and compare the samples.
Looking forward to your suggestions! Many thanks.