Looking for a protocol for DiI staining. Lower concentrations of PFA seem preferable with vibratome sectioning. Is there a preferable way to deliver crystals to the hippocampus or brain in general? Is using a micropipette enough?
Iontophoresis using a micropipette: http://www.ncbi.nlm.nih.gov/pubmed/7743937 ; you will need a pipette holder with internal silver wire electrode and side port for this. I use this kind of device for labelling DRG sensory neurons in 250 micron-thick transverse sections of E6 chick or E14 mouse embryos. Embryos are fixed overnight at 4oC in 4% PFA/PBS before cutting on Vibratome.
Or, if your specimen is large and your target area easily accessible, you can pick up DiI crystals with sharpened tweezers or tungsten wire and place them directly onto the tissue.
You can also have a look on this Jove protocol, to get some more tips:
http://www.jove.com/video/3667/dii-labeling-drg-neurons-to-study-axonal-branching-whole-mount
Good luck!
I am using a gene gun to shoot microDiI crystals to specific brain areas ( the protocol is called biolistic staining). You can look at the protocol in: L. Enriquez-Barreto, G. Cuesto, N. Dominguez-Iturza et al., “Learning improvement after PI3K activation correlates with de novo formation of functional small spines,” Frontiers in
Molecular Neuroscience, vol. 6, article 54, 2014. Sorry for the self –promotion. In the article (Figure 9) there is also a picture of pyramidal CA1 neuron with the staining you can get.
Best
Thank you Miguel. We do not have a gene-gun, so I am using my good fine motor skills to deliver crystals with a needle under a microscope.
Hi Tatyana...
Just follow your question.
Have succeeded doing DiI staining?
I am still on going to looking the best way for this method. I think this method is slightly easy and fast than golgi staining.
I had tried rapid golgi staining method and the result was unsatisfied for me.
Thus, I switch to DiI staining. however, my lab doesnt have gene gun too. We use fine needle to apply the crystal. Mostly result show clumping crystal on the brain slice (P12 sample), after I observed under confocal microscope, dendritic shaft show artefact (look like buble along the shaft).
So, really hope some advices from you or other for doing this DiI staining.
Thanks
Ria
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