DiI labeling and fixation

I’ve been trying to retrogradely label DRG neurons from various sites. All the labeling methods have their advantages and drawbacks.

DiI is commonly used. I have a 5 mg/ml stock in DMSO. I’ve noticed that it’s kind of ‘messy’ at the DRG. Not just in soma but also in some surrounding cells. It’s difficult to tell what’s truly positive.

I’ve heard that DiI is not fixable. Is this true? I fixed these and these have been treated with triton-containing buffer. I’ve also heard to not use detergents.

See this discussion for fixation: https://www.researchgate.net/post/Do-GFP-or-DiI-leak-from-cells-following-fixation-with-4-PFA-What-rate-will-they-be-lost

Is this typical DiI staining?

I ultimately plan to do FACS, so the fixation stuff might not be an issue.

The benefit of DiI is its abundant and much cheaper than Fast Blue and red fluorescence is available on most machines, while FB is UV/blue.

@tberta @thicunha @SamineniV @sshiers @defaria

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Hi @achamess. I have no experience with Dil. Have you tried AAV-Cre in flox-reported mice? It could be one alternative. Please see this paper in which von Adrian labeled sensory neurons innervating the lymph nodes (Lymph nodes are innervated by a unique population of sensory neurons with immunomodulatory potential - ScienceDirect) our this author used for joints (https://onlinelibrary.wiley.com/doi/10.1002/art.41314).

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Thanks @thicunha . We have tried various AAV-Cre in Ai9 or Ai32, with variable success. The best so far is AAV1-Cre, which is what they used in the von Adrian paper. Here is an AAV1-Cre into Ai32 hindpaw

Pretty good. Probably not as much as the dyes though.

Great. how much did you inject? Temo is injecting in newborn, and the results seem very good.

5 ul of 10^13. So that’s pretty high. Undiluted. I know Temo is getting great results in younger animals. But we need adults and also I need to make sure its specific and not spreading outside the projection territory, which is something I think @tberta has said is an issue with the young injections.

This point is really difficult to control, even with dyes. I guess Temo is injecting 10 microL. So, maybe injecting less 1-2 microL (intradermally, not subcutaneously) and look at the contralateral DRGs. In the Brain, we started injecting 200 nL, and now we realize that 25 nL is enough.

We inject 5 ul of AAV9-syn-cre subcutaneously in the glabrous territory of the paw of P10 mice, and it works fine but it still spreads to DRG neurons outside the territory (i.e. cervical DRG neurons, or contralateral DRG neurons). Small intradermal injections is an idea worth considering.

Retrograde tracers can be an alternative. Unfortunately, the best retrograde ones are FastBlue and FuoroGold. DiI is messy and FluoroRuby is a little better but not great.

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@dmolliver Do you have any thoughts on this? Someone mentioned WGA on Twitter

Any concern for biased labeling, like you mentioned previously about CTB?

Hey, @achamess have you ever tried to do block (PBS/BSA) your cells before using the DiI? When I’m working with this dye, it makes all the difference

Hi @castellirf - What do you mena block cells before DiI? I’m injecting DiI into live mice in various locations and then taking DRGs for cryosectioning and IHC. How and when would I block with BSA?