I'm trying to stain ~ 1 mm^3 volumes of PFA-fixed brain tissue with nanoparticle-antibody conjugates. Although my nanoparticles are 5 nm in diameter, I'm experiencing difficulty in getting the particles to diffuse into the tissue. What techniques might help make the tissue more permeable to these constructs? It looks like tissue clearing methods (i.e. CLARITY, CUBIC, etc.) might be helpful, does anyone have recommendations around those?
Hello,
Clearing methods like CLARITY replace lipids by hydrogel, so removing the membrane obstacle for antibody penetration. Then immunolabelling can be done but you will need several days/a week with antibody incubation, even DAPI staining requires 3 days incubation.
If you want to see deep inside your tissue piece, you will then need special mounting medium and objective lens. Mounting medium and objective lens will vary depending on which clearing methods you will use.
I first suggest to contact imaging facilities around your location to see which equipments and methods they can propose, which lab is doing these methods.
Hi Logan,
Have you considered using reagents that (partially) solubilize membranes in the cells? Triton x-100, NP-40 and saponin are much used for this. I don't know how they perform in brain tissue.
Good luck
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