Hi Research Community,
I am looking for a method to label dendritic spines in fixed hippocampal brain slices.
Due to our fixation Golgi staining seems to be off the table.
Now I found Dil labelling as an alternative, but most labs use gene guns, that I unfortunately don't have. Has anyone experience with using dye crystals added directly on the tissue or alternatives?
Thanks
Luisa
Hi Luisa,
I have had experience with this recently using the green carbocyanine version (DiO) in fixed mouse brain slices (250um thickness) & found the following resources very helpful:
https://protocols.scienceexchange.com/protocols/dendritic-spines-observed-by-extracellular-dii-dye-and-immunolabeling-under-confocal-microscopy
Article Acute slice preparation for electrophysiology increases spin...
It can be a bit tricky to optimise as it requires some quite fine motor skills but essentially:
- Tissue should be fixed with 1.5% PFA (if you go to 4% you might really struggle to get effective dye diffusion & I would avoid heating antigen retrieval protocols also)
- Sonicate DiI/DiO overnight in an eppendorf to make the crystals into a finer powder
- Place tissue onto flat surface and aspirate and solution off the top (the DiI will not easily stick to the tissue/will come off the applicator if it passes through solution)
- Apply small DiI crystals to the hippocampal cell layer using a thin tungsten needle or glass micropipette - gently touch above the cell layer and allow the crystal to adhere to the tissue to avoid damaging the sample
- Tricky part is balancing crystal size and placement (I think the above publication estimated 20um size). Position crystals generally with at least 200um distance between them to reduce the risk of cell staining overlap
- Rehydrate the section in small volume of 1X PBS (~20-50ul) and cover - I found using the method from Trivino-Paredes et al. (2019) worked best: cover slice in PBS with parafilm to apply pressure (typically I put parafilm on top and underneath the slice making a 'sealed bubble').
- Incubate at RT for 24-hours without shaking (may want to test 4C vs RT and experiment with duration but 24h at 4C was optimal for my conditions)
- Carefully remove the parafilm from the section and place on your slide - check staining quality
I have successfully combined this with IF, performing the DiO labelling first, then performing the IF free-floating. Generally the hardest part is optimising the practicality of placement/size of the crystals. Sonicating them for longer can help and using a very fine tipped applicator I found worked best.
Good luck!
Couple of questions:
Are you sonicating the dye (DiI/DiO) while its in solution?
Do you have any tips and tricks for getting the crystal onto the tungsten needle? What is your experimental setup at this step?
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