The second characterization approach is based on a modified protocol from Campanella and collaborators 9 that provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry. This method is a very powerful way to characterize numbers, dynamics and phenotypic changes of infiltrated neutrophil subpopulations of the ischemic tissue 8. This is the best choice for cell quantification when the tissue is cut into sections, as it avoids the bias on cell estimation because of the fragmentation of the tissue into sections. This is the most commonly used stereological probe in life sciences and provides a high degree of precision with low coefficients of error 5-7.
The first approach to characterize myeloid cell subsets of the ischemic brain is a stereological method based on the optical fractionator approach 5.
#CAVALIERI COEFFICIENT OF ERROR FOR STEREOLOGY HOW TO#
In addition, it also provides a description of an experimental model of cerebral ischemia in mice, which consists of the transient or permanent ligation of both distal Middle Cerebral Artery (MCA) and the common carotid artery (CCA), including how to evaluate the infarct by the accurate Cavalieri method using a stereological software. This protocol provides two approaches to estimate the number of different myeloid cell subsets of the ischemic brain and to perform their phenotypical characterization. Morphological, phenotypic and gene expression alterations of microglia, as well as extravasation and activation of haematogenous macrophages and neutrophils are believed to play a pivotal role in the pathophysiological cascade following brain injury 1-4. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke.
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