One of the tasks in my project is to immunostain for tuj1 and cfos in whole mount colon section of E18.5 mice. Since the tissue is quite small and fragile we dont open it longitudinally, but rather place it as is on the slide (after completing the immunostaining process). Additionally, we put nail polish around the slide before placing the coverslip so as to account for the height of the tissue itself.

However, we've consistently encountered the problem where 1-2 out of 6 tissues comes out okay, with clear tuj1 and cFos staining, whilst the rest either have sub-par tuj1 or cFos staining (as seen in attached example images). We use the same conditions of fixation, permeabiliziation as well as even being placed on the same slide. We've even tried different whole mount protocols with slightly varying fixation/permeabilization times as well as RT vs 4c, to no avail.

Side note: with Tuj1 we expect to see a web like network of the ENS and with cFos, we expect to see primarily as localization to the nucleus.

Has anyone encountered something similar and what ended up being the problem? If not, are there any recommendations for culprits/factors that could explain this variability?