🚀 go-pugleaf

I don't think these methods are new, but for those that are interested,

I label whole invertebrate larvae with antibodies against 5-HT and a
peptide call SCP. These larvae are about 200 X 70 um so their they're
effectively thicker than your sections. There are two methods that work
to get the antibodies into the tissues.

1. Dehydrate the tissue through and alcohol series (30%, 50%, 70%, 80%,
90%, 100%) leaving the tissue in each solution for 5 - 20 min, then
rehydrate back to water through the same series. I've also gone through
a number of 100% EtOH changes and then into toluene or xylene and then
back down the series to water. Presumably this removes lipid from the
cell membranes and makes them more permeable. It's possible it also makes
small holes in the membranes. At any rate, it does permeabilize the tissue.

2. The second method is less drastic. Soak the tissue in PBS containing
5% (yes, that's 5%, not 0.5%) Triton X-100. The length of the soaking
period for my larvae is 1 - 3 days (in the refrigerator). This also
permeabilizes the tissue. I've had some indication that if the soaking
period is too long, it may have a negative effect on the labeling. This
method seems to give me the best results for the neurons and axons I'm
looking at.

After either of these treatments, rinse the tissue in your normal PBS
solution and procede with your labeling using the reagents you normally use.

Good luck!

Steve Kempf

kempfsc@mail.auburn.edu