I am having some difficulties with my stainings in the mouse spinal cord. I am using longitudinal, both vibratome and cryo-, 4%PFA-fixed sections. The problem is that, with protocols I have tried so far, using longitudinal sections results in a non-uniform staining pattern. It works pretty fine in the grey matter, but the penetration in the white matter axons is pretty low. I am trying to work this out by using tissue from transgenic mouse lines with fluorescently labelled neurons and neuronal mitochondria and anti-GFP antibody. I have tried X-100 Triton (0.1-1%) permeabilization and several fixatives - methanol, methanol+acetone, ethanol-acetic acid. It helps with the axons, but it is still hard to get the antibody into mitochondria.
Any feedback would be greatly appreciated !
Thanks a lot in advance,
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