I want to use DiI (or other dyes that work in postmortem tissue) to trace inputs to the striatum in adult mouse brains (mainly cortico-striatal and thalamo-striatal circuits). The brains have been perfused with 4% PFA and have been kept in 1X PBS with 0.02% sodium azide for a few months at 4C. Is it still possible to use DiI to trace neural circuits? If so, does anyone have a recommended protocol I could use?
Thank you!
Hi Andrew,
Yes, you can still get high-quality DiI staining with tissue that has been preserved @ 4°c for several months! I’ve been able to successfully image dendritic spines from sliced tissue that was stored for 6-months in 1.5% PFA 1x PBS @ 4°C.
Although I will say the tissue was a bit harder to work with as it got older, however, at the microscopic level, the neuronal morphology was still fully preserved.
But I imagine because you’ve used 4% PFA instead, it’s probably still fine for even longer periods.
I’m not sure if you’ve already sliced the tissue, but I recommend using a vibratome (or manual slicing) rather than a cyrotome/microtome when using lipophilic dyes. This is because freezing the tissue will disrupt the neuronal membrane to which the lipophilic diI needs to adhere. In our experience, the diI staining becomes very hazy and blurry if the tissue has been frozen before. However, this might still be fine with neuronal tracing (but just not for visualizing fine-grained components like dendritic spines).
As for general protocols, a good starting point is this one from Rasio-Filho et al., 2010 and one from Trivino-Paredes et al., 2019
Article NATURE PROTOCOL EXCHANGE - Dendritic spines observed by extr...
Article Acute slice preparation for electrophysiology increases spin...
Best of luck!
Stephanie
Stephanie Jane Huang Thank you so much for your response! This is very helpful. Brain are still intact, so when I do my experiment I will make sure to use a vibratome.
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