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


#21

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.


#22

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


#23

Mostly just to save some money…sorry ACD!


#24

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!


#25

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.


#26

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.


#27

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


#28

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.


#29

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.


#30

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.


#31

Hey !
Did it work with RNAscope ?
Thanks


#32

Unfortunately not. The FB washed out.


#33

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


#34

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