Ruffled sections

[katiec]

Hi, I am performing free floating immunohistochemistry with mouse brain sections.  My protocol says to finish with two washes in distilled water and then to mount the sections on the slides in distilled water.  However, when I wash the sections in distilled water they shrink and ruffle up.  I get that this probably has something to do with osmosis but why is it happening? 

The mice were cardiac perfused with 4% paraformaldehyde and the brains postfixed for two days then spent three days in sucrose (over the weekend).  I sectioned on a vibrotome and labeled with primary antibody (NeuN) yesterday and secondary (RbxMs, Cy5) today.

[richard03]

You need to add a few drop of Tween 20 to reduce surface tension. Give it a try and it should work fine.

[katiec]

Great, thank you!  I will try that.  Another colleague also suggested using 50 mM Tris-HCl instead of water.  Would you agree that could work?

[richard03]

PBS or TBS with 0.05% Tween 20 will be best!

[excalibur]

The salts in PBS will precipitate out after the water has dried and form crystals on the slide.