Because the unbiased nature of the Fractionator approach depends on whether we sample the same percentage of the tissue for each section (not the total amount of tissue sampled), you’ll still achieve an unbiased estimate as long as you set up the Optical Fractionator using the same parameters for each section of the same subject. Therefore, it’s actually expected that, in most cases, the number of counting frames will vary from section to section; because there is a (systematic) random placement of the disectors and the size and shape of the cross-section of any given region will change as the microtome proceeds through the tissue, the number of counting frames should change accordingly in order to ensure that the same percentage of tissue is sampled for each section.
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!