Hey everyone,
I've been working with GRIN lenses (Inscopix) for almost 1 year and still had no luck seeing strong fluorescence on them. When I look at them, I can see good blood flow and, sometimes, some fluorescence, but never enough to get significant conclusions.
I've tried different virus concentrations (currently we're using 1:6), intervals between virus injection and lens implant (2-4 weeks), checking the calcium signal after 4-6 weeks, and still, I can't see a nice fluorescence.
It's important to highlight that when I perfuse the animals to check the virus expression, it is not as high as other regions/mice strains (since we depend on the D1 expression in the mPFC) but I'm still able to mark and visualize the GFP neurons in the confocal.
I've also injected in the striatum since it's another region that is involved in the type of behavior that I study, but I haven't done this immuno yet (probably in the following days).
In light of everything mentioned, does anyone have any suggestions to improve the fluorescence? Maybe some tips for the surgeries, intervals, virus, regions, concentrations etc. Every suggestion is more than welcome.
I'm attaching an example of one animal that I checked the fluorescence.
Let me know if you need further information and thank you very much for the help.
P.S. Relevant information:
- Target region: medial prefrontal cortex (mPFC) - DV -2.3mm
- Lens: Proview Integrated lens 0.5x4.0mm Inscopix
- Mice strain: D1-cre
- Virus: pAAV.Syn.GCaMP6f.WPRE.SV40 (Plasmid #100837 - Addgene)
- System: nVista Inscopix.
1. Be very careful with blood. Blood is the enemy of your imaging. Always clean the implantation surface site with saline. When you insert the GRIN lens, there should be no blood on the way. Use absorption tips to help you with that.
2. The field of view you use when imaging should be narrow. Try to avoid having the boundaries of the GRIN lens—it will be harder later with the analysis steps.
3. Consider using the Ai95 (or similar) to cross with your D1-Cre. It will be much more efficient.
4. You can use the 0.6mm GRIN lens, which will increase your chances of getting a signal.
5. Two more points about the surgery: Some people aspirate or use a needle to create a 'track' for the GRIN lens. That might help. In any case- insert it very slowly, to prevent damage to the tissue.
Hey Anat, thanks for the quick reply. Please find below my comments:
1. Yes, I always clean the surface with cold saline during the procedure and avoid blood. I only start the lens implant when there is no blood around.
2. Thanks for the reminder, I'll try harder to improve the placement to avoid the boundaries.
3. I'm not familiar with this strain. I'll look for more information (although I don't think it's feasible at the moment).
4. We contacted the Inscopix support before choosing the best dimension and the 0.5mm was their suggestion (since we also had the 1.0mm), but I'll consider the 0.6mm as well.
5. Yeah, here we use a needle to track the path before the implant and we do it very slowly and gently.
Once again, thank you for the help.
Regards.
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