Stereology methods require tissue to be cut in thick sections (such as 50 or 60um in thickness), but it becomes a challenge for immunohistochemistry, as thick sections can be hard for antibodies to penetrate. I recently started cutting the rodent brain in 60um, and noticed that there was substantial variation in the mounted section thickness (between 17 and 25um) after H&E staining (i.e. EtOH & xylene cycles) of different batch. I assume that it was mainly due to the variation in shrinkage by alcohol dehydration during the processing. I wonder if anyone had looked into the possibility to reduce tissue shrinkage, so the tissue could be cut thinner for optimal immunostaining? If not using any alcohol in the process, but only air-drying after mounted from free-floating immunofluorescent staining and using water-based coverslip medium, would it result in less shrinkage? What would be the minimal thickness for cutting to use optical fractionator for immunofluorescent markers? Thank you.
the the best way to prevent the brain shrinkage is to use phosphate buffer formalin as fixative. the second step is to leave the tissue in oven 55 c for three successive hours hour, change the wax after every hour. these two most important steps
In my hands its hard to avoid alterations due fixation and or dehydration.
The best way I found to bypass this issue is using confocal microscopy. Sampling images to a fixed thickness in the range area where fluorescence signal can be observed.
- Badges
- Science method