Abstract
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.
Highlights
The introduction of stereological methods to neuroscientific questions has provided novel and, more importantly, reliable tools for quantitative data collection, rapidly becoming the gold standard in the field
As a test of our protocol, we present an optical fractionator study of rat cortex at postnatal day 10 that estimates: (1) the number of subcerebral projection neurons, defined by co-labeling of the established markers Ctip2 and NeuN (Arlotta et al, 2005; Lyck et al, 2007), and (2) the total population of NeuN+ neurons
Attesting to the efficacy of our protocol, we found that it facilitated the unambiguous identification of Ctip2+/NeuN+ subcortical projection neurons, yielded low error rates, and produced estimates of NeuN+ neurons consistent with published data, even in a small sample of animals (N = 5)
Summary
The introduction of stereological methods to neuroscientific questions has provided novel and, more importantly, reliable tools for quantitative data collection, rapidly becoming the gold standard in the field. An obvious additional advantage of multiple labeling is the ability to quantify multiple cell types in one tissue series or even stereological sampling run In rare cases, this can be accomplished with one antibody. Multiple chromogens can be used in enzymatic techniques, fluorophores emitting in individual fluorescence channels provide more distinct and consistent labeling, improving discrimination between antibodies This helps to ensure that the correct cell types are sampled during data collection. Some researchers favor enzymatic immunohistochemistry for stereology as the labeling is maintained nearly indefinitely To overcome these challenges, we have tested several experimental procedures and fluorophores to develop a reliable working protocol for the stereological quantification of diverse cell types using immunofluorescence.
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