Design-Based Stereology for Evaluation of Histological Parameters
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Valid quantification of organ volume and total cell numbers are crucial parameters for morphometric studies. The number of a specific cell type cannot be simply deduced from the number of its profiles found in thin tissue sections, as this parameter also depends on cell volume, tissue orientation as well as tissue atrophy. Design-based stereology has become the method of choice for unbiased, reproducible total cell number quantification. Steps described in this protocol include transcardial perfusion of mice, postfixation, and cryoprotection of the region of interest (ROI), followed by the preparation of a systematically and randomly sampled series of thick sections through the entire ROI. Furthermore, it is described how to perform immuno-histochemical staining of such thick cryo-sections, followed by providing a guidance for quantification of the ROI volume, the generation of unbiased virtual counting spaces, and steps to work with these counting spaces to obtain an unbiased estimate of total cell numbers.
KeywordsStereology Cell number Volume Quantification Unbiased Systematic
We thank Helga Helten and Astrid Baltruschat for administrative support during the writing of this protocol.
Markus Kipp and Maren Kiessling contributed equally to the work being described. Markus Kipp and Maren Kiessling wrote the protocol. Tanja Hochstrasser and Caroline Roggenkamp validated the protocol. Christoph Schmitz did supervise the protocol and commented on the manuscript at all stages.
Compliance with Ethical Standards
Competing Financial Interests
This protocol was supported by SRC ProMyelo (MK). No financial support by third parties was obtained, and no benefit was received or will be received directly or indirectly from a commercial party related to the work being described. Christoph Schmitz serves as paid consultant for and receives benefits from MBF Bioscience (Williston, USA), the provider of the Stereo Investigator software. However, Christoph Schmitz has not received any honoraria or consultancy fee in the context of the work being described. Markus Kipp, Maren Kiessling, Tanja Hochstrasser, and Caroline Roggenkamp declare that they have no competing financial interests.
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