I am trying to stain regenerating axons with an antibody in a whole mount sciatic nerve preparation but the antibody does not seem to be properly penetrating the tissue.
Whether frozen or paraffin-embedded sections, Tuj1 (Anti-beta III Tubulin) antibody works well to better understand the the degree of which peripheral nerves with axons undergo regeneration after Wallerian degeneration. Further, antibodies recognizing NGF receptor p75 or schawann cell marker GFAP are recommended.
As shown in the attached file, after sciatic denervation Dct-H2B-GFP+/TuJ1+ cells disappeared from the dermis, suggesting that the dermal GFP+ cells are peripheral neurons or associated neuron-dependent precursor cells.
What are your incubation times? I work with floating sections of 50um and need overnight incubation with primary antibody. A whole, unsectioned nerve may need more time. Also, during the blocking step, are you using Triton-x 100? This serves to permeabilise the tissue a bit helping the antibody get through it.
Try DSMO treatments in advance, let Ab better penetrate, don't forget to wash DSMO out.
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