We have located links that may give you full text access.
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 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.
Full text links
Related Resources
Trending Papers
Challenges in Septic Shock: From New Hemodynamics to Blood Purification Therapies.Journal of Personalized Medicine 2024 Februrary 4
Molecular Targets of Novel Therapeutics for Diabetic Kidney Disease: A New Era of Nephroprotection.International Journal of Molecular Sciences 2024 April 4
The 'Ten Commandments' for the 2023 European Society of Cardiology guidelines for the management of endocarditis.European Heart Journal 2024 April 18
A Guide to the Use of Vasopressors and Inotropes for Patients in Shock.Journal of Intensive Care Medicine 2024 April 14
Diagnosis and Management of Cardiac Sarcoidosis: A Scientific Statement From the American Heart Association.Circulation 2024 April 19
Essential thrombocythaemia: A contemporary approach with new drugs on the horizon.British Journal of Haematology 2024 April 9
Get seemless 1-tap access through your institution/university
For the best experience, use the Read mobile app
All material on this website is protected by copyright, Copyright © 1994-2024 by WebMD LLC.
This website also contains material copyrighted by 3rd parties.
By using this service, you agree to our terms of use and privacy policy.
Your Privacy Choices
You can now claim free CME credits for this literature searchClaim now
Get seemless 1-tap access through your institution/university
For the best experience, use the Read mobile app