Stuff I tried during grad school/post-doc that won't get published but could be useful

Along the same lines, I have successfully diluted probes (in ACD provided diluant). Started with just channel 2/3 as you must dilute them anyway, but can also be done with C1.

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Interesting! What was your reason for diluting? To use less or to bring down strong signal? Either way, cool!

Mostly just to save some money…sorry ACD!

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Cool stuff! I’m just curious what you are using to get blue light to the paw? Would a cheap and cheerful blue laser pen be good enough to get chr2 in nerve endings? Thanks a lot!

Thanks. Fiber-coupled LED from Thor Labs.

https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=5206

You need to get at least 1 mw/mm^2 of light, so if you can get a LED pen that can do that, you can try it out. But you won’t have control on parameters like pulse-width, frequency, etc. The Thor system is pretty affordable. They’ve even got a very no frills starter kit:

https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=6148

We use the DC2200 driver. I like it.

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A follow up. AAV1-hSyn-Cre from Penn. 1^13 vg/ml (10 ul) into the plantar hindpaw. Here is the spinal cord from this mouse at 4 weeks. Mostly very superficial DH with some deeper. I may have gotten a proprioceptor as well. Will need to use nuclear fluorophores to assess anterograde spread.

Hey! We are going to pull the trigger and try out Neurotrace, are you using it with RNAscope?

Awesome!It doesn’t look as good after RNAscope, presumably because of protease chewing up ribosomal proteins? Try 1:200 for 10 minutes. Extend the time if you want it darker. It’s not perfect after RNAscope but it’s not bad.

Translating Ribosome Affinity Purification (TRAP) in various DRG neurons
Back in 2014-2015, I was keen on trying to get TRAP to work in DRG. I tried with TRPV1-Cre and LSL-Rpl10a-EGFP mouse from Jax. There was labeling of the neurons, but the pulldowns for mRNA brought down a lot of nonspecific RNA and it was hard to see enrichment. Speaking to various other researchers who’ve used TRAP, this seems to be a common thread. So I abandoned the method. I do know that one group in the pain field has gotten TRAP to work on DRGs, so it’s not impossible. But it requires a lot of optimization.

Retrobeads work well as a retrograde tracer from the plantar hindpaw.
2.5 days after injection. 5 ul. I could probably afford to reduce the volume. That’s a lot of labeling.

I want to see if this holds up to RNAscope. Fast Blue washes out.

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Hey !
Did it work with RNAscope ?
Thanks

Unfortunately not. The FB washed out.

Thank you for your answer, I meant retrobeads combined with RNAscope, did you try ?

I didn’t try RNAscope. But you should! :slight_smile: and let us know how it goes.