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Maximizing Explanatory Power in Stereological Data Collection: A Protocol for Reliably Integrating Optical Fractionator and Multiple Immunofluorescence Techniques

With the promise of greater reliability and replicability of estimates, stereological techniques have revolutionized data collection in the neurosciences. At the same time, improvements in immunohistochemistry and fluorescence imaging technologies have facilitated easy application of immunofluoresce...

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Autores principales: Kreutz, Anna, Barger, Nicole
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218486/
https://www.ncbi.nlm.nih.gov/pubmed/30425623
http://dx.doi.org/10.3389/fnana.2018.00073
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author Kreutz, Anna
Barger, Nicole
author_facet Kreutz, Anna
Barger, Nicole
author_sort Kreutz, Anna
collection PubMed
description With the promise of greater reliability and replicability of estimates, stereological techniques have revolutionized data collection in the neurosciences. At the same time, improvements in immunohistochemistry and fluorescence imaging technologies have facilitated easy application of immunofluorescence protocols, allowing for isolation of multiple target proteins in one tissue sample. Combining multiple immunofluorescence labeling with stereological data collection can provide a powerful tool to maximize explanatory power and efficiency, while minimizing tissue use. Multiple cell classes, subtypes of larger populations, or different cell states can be quantified in one case and even in one sampling run. Here, we present a protocol integrating stereological data collection and multiple immunofluorescence using commonly employed widefield epifluorescence filter sets, optimized for blue (DAPI), green (FITC), and far red (CY5) channels. Our stereological protocol has been designed to accommodate the challenges of fluorescence imaging to overcome limitations like fixed filter sets, photobleaching, and uneven immunolabeling. To enhance fluorescence signal for stereological sampling, our immunolabeling protocol utilizes both high temperature antigen retrieval to improve primary antibody binding and secondary antibodies conjugated to optimally stable fluorophores. To illustrate the utility of this approach, we estimated the number of Ctip2 immunoreactive subcerebral projection neurons and NeuN immunoreactive neurons in rat cerebral cortex at postnatal day 10. We used DAPI (blue) to define the neocortex, anti-NeuN (far red) to identify neurons, and co-labeling of anti-Ctip2 (green) and anti-NeuN (far red) to isolate only subcerebral projection neurons. Our protocol resulted in estimates with low sampling error (CE < 0.05) and high intrarater reliability (ICC > 0.98) that fall within the range of published values, attesting to its efficacy. We show our immunofluorescence techniques can be used to reliably identify other cell types, e.g., different glial cell classes, to highlight the broader applications of our approach. The flexibility of the technique, increasingly reduced costs of fluorescence technologies, and savings in experimental time and tissue use make this approach valuable for neuroscientists interested in incorporating stereology to ask precise neurophysiological and neuroanatomical questions.
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spelling pubmed-62184862018-11-13 Maximizing Explanatory Power in Stereological Data Collection: A Protocol for Reliably Integrating Optical Fractionator and Multiple Immunofluorescence Techniques Kreutz, Anna Barger, Nicole Front Neuroanat Neuroanatomy With the promise of greater reliability and replicability of estimates, stereological techniques have revolutionized data collection in the neurosciences. At the same time, improvements in immunohistochemistry and fluorescence imaging technologies have facilitated easy application of immunofluorescence protocols, allowing for isolation of multiple target proteins in one tissue sample. Combining multiple immunofluorescence labeling with stereological data collection can provide a powerful tool to maximize explanatory power and efficiency, while minimizing tissue use. Multiple cell classes, subtypes of larger populations, or different cell states can be quantified in one case and even in one sampling run. Here, we present a protocol integrating stereological data collection and multiple immunofluorescence using commonly employed widefield epifluorescence filter sets, optimized for blue (DAPI), green (FITC), and far red (CY5) channels. Our stereological protocol has been designed to accommodate the challenges of fluorescence imaging to overcome limitations like fixed filter sets, photobleaching, and uneven immunolabeling. To enhance fluorescence signal for stereological sampling, our immunolabeling protocol utilizes both high temperature antigen retrieval to improve primary antibody binding and secondary antibodies conjugated to optimally stable fluorophores. To illustrate the utility of this approach, we estimated the number of Ctip2 immunoreactive subcerebral projection neurons and NeuN immunoreactive neurons in rat cerebral cortex at postnatal day 10. We used DAPI (blue) to define the neocortex, anti-NeuN (far red) to identify neurons, and co-labeling of anti-Ctip2 (green) and anti-NeuN (far red) to isolate only subcerebral projection neurons. Our protocol resulted in estimates with low sampling error (CE < 0.05) and high intrarater reliability (ICC > 0.98) that fall within the range of published values, attesting to its efficacy. We show our immunofluorescence techniques can be used to reliably identify other cell types, e.g., different glial cell classes, to highlight the broader applications of our approach. The flexibility of the technique, increasingly reduced costs of fluorescence technologies, and savings in experimental time and tissue use make this approach valuable for neuroscientists interested in incorporating stereology to ask precise neurophysiological and neuroanatomical questions. Frontiers Media S.A. 2018-10-30 /pmc/articles/PMC6218486/ /pubmed/30425623 http://dx.doi.org/10.3389/fnana.2018.00073 Text en Copyright © 2018 Kreutz and Barger. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Neuroanatomy
Kreutz, Anna
Barger, Nicole
Maximizing Explanatory Power in Stereological Data Collection: A Protocol for Reliably Integrating Optical Fractionator and Multiple Immunofluorescence Techniques
title Maximizing Explanatory Power in Stereological Data Collection: A Protocol for Reliably Integrating Optical Fractionator and Multiple Immunofluorescence Techniques
title_full Maximizing Explanatory Power in Stereological Data Collection: A Protocol for Reliably Integrating Optical Fractionator and Multiple Immunofluorescence Techniques
title_fullStr Maximizing Explanatory Power in Stereological Data Collection: A Protocol for Reliably Integrating Optical Fractionator and Multiple Immunofluorescence Techniques
title_full_unstemmed Maximizing Explanatory Power in Stereological Data Collection: A Protocol for Reliably Integrating Optical Fractionator and Multiple Immunofluorescence Techniques
title_short Maximizing Explanatory Power in Stereological Data Collection: A Protocol for Reliably Integrating Optical Fractionator and Multiple Immunofluorescence Techniques
title_sort maximizing explanatory power in stereological data collection: a protocol for reliably integrating optical fractionator and multiple immunofluorescence techniques
topic Neuroanatomy
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218486/
https://www.ncbi.nlm.nih.gov/pubmed/30425623
http://dx.doi.org/10.3389/fnana.2018.00073
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