Sectioned tissue is not perfectly flat. Think of the surface as a landscape covered with hills and valleys. To identify the top of your tissue, use a high power objective (with a high numerical aperture) to focus up until you are out of focus above the tissue. Slowly focus down until you can see the top of the ‘hills’ come into focus, that is, until only the highest parts of the tissue surface are in focus. Note this Z position. This is the very top of the tissue.
This FAQ comes from stereology.info, our curated collection of information and references regarding the use of stereology in the biological sciences. Feel free to review the other FAQs there as well. We hope this thread will spark some in-depth discussion on stereology!