Hi everyone, I’m new to pain research and would like to try whole mount stain for murine DRG tissue.
I’m wondering, does the DRG require tissue clearing? If so, any tips for how to physically handle a tissue-cleared DRG? What should I perform the clearing in and how do I transfer this to a slide for imaging?
Thank you!
Hi, sorry I have never performed whole mount staining before on mouse DRG. I’ve been clearing human DRGs, but I don’t have any protocols optimized yet.
We did not clear the DRGs, but the wholemount can be visualized in regular confocal microscopy.
You can have this kind of image: Red Nav1.8Tdtomato and Green-Cx3cr1 (both in transgenic background)
But, @Niels Eijkelkamp did clearing in this paper: https://www.sciencedirect.com/science/article/pii/S0896627321009697?via%3Dihub
Thank you so much everyone. I read here (https://www.sciencedirect.com/science/article/pii/S0165027022000243) a trick to identify tissue-cleared DRG by using a weak UV light to locate a DAPI signal - it worked well.
This is a great protocol reference from Tony Yaksh’s group (PMID: 35181343). In our histology and imaging core we are moving towards imaging whole cleared DRG for multiplex IHC and it works quite well with many but not all antibodies. Even with a simple clearing protocol like BABB you can get surprisingly good results, enough that I am wondering if we should ever use tissues sections for colocalization studies any more. Attached is a widefield single field unprocessed image of whole mount DRG IHC with BABB clearing at 10x that took about a week to process, so you can imagine how good it could be with some optimization and confocal (TRPV1 in green, TH in red).
This looks beautiful! Thank you so much, I will definitely give this protocol a try. I agree, I think whole mount is more suitable for colocalization studies versus sections.
Beautiful! What kind of imaging are you doing? Confocal or light sheet?
I agree, if one can circumvent the need to section, you’ll get better quantification and possibly easier/less complicated experiemental setups.
The above is just standard widefield epifluorescence, so it’s the least you can do in terms of processing or advanced imaging. I am attaching a single optical confocal plane from another whole mount processed by our Histology and Imaging Core. I think the take-away is just how much you can achieve without any fancy equipment, any lab can get the image above, and if you go to confocal you can get some truly outstanding images and 3D reconstructions.
