Feb 02, 2023
Stereologic Cell Population Estimates in Cleared Human Brain Tissue
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Bridget Wicinski, Patrick.hof 2023. Stereologic Cell Population Estimates in Cleared Human Brain Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l273dqg1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 27, 2023
Last Modified: February 02, 2023
Protocol Integer ID: 75991
Funders Acknowledgement:
Patrick Hof
Grant ID: U01 MH117023-01
Abstract
We provide a step-by-step protocol for stereologic assessment in the human cerebral cortex using cleared postmortem specimens with a laminar level of analysis for distinct neuronal populations identified by immunolabeling for specific cytoplasmic markers. Stereologic tools provide a set of simple rules and formulas to estimate numerical quantities of various morphologic parameters in tissue, with precision, accuracy and a design free of observer-induced bias. Parameters such a number, density, volume, surface area, or length can be easily estimated based on the systematic sampling of a region of interest (e.g., a layer of a cytoarchitecturally defined region of cortex, defined as a “volume of reference”), with an observer-independent random design, that gives each object under study (e.g., a neuronal population identified by a specific protein marker) the same probability to be sampled once and only once in its volume of reference, based on strict sampling criteria that are kept constant for a given object throughout the analysis. Stereologic analysis was performed on cleared slice from large specimens encompassing Broca’s area (Brodmann’s area 44/45), up to 4x4x2 cm, imaged at 3x3x3 µm pixel dimensions, and immunostained for NeuN, calretinin, and somatostatin, using the MBF Bioscience Stereo Investigator Cleared Tissue software (version 2022.1.1) with an Optical Fractionator design. Data were recorded as estimates of total number of neurons as well as neuronal densities in each layer. The protocol provides the detail of the sampling parameters including counting frame size, grid size, and disector height, definition and contouring of cortical layers and use of their boundaries to estimate laminar surface areas and volume. Full details on software operation and sampling design are explained.
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