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Microglia in Human Postmortem Brain Samples: Quantitative Ultrastructural Analysis of Scanning Electron Microscopy
Marie-Kim St-Pierre1,2,3, Eva Šimončičová3,4, Micaël Carrier1,2,3
1Axe neurosciences, Centre de recherche du CHU de Québec-Université Laval, Québec, QC, Canada.
Abstract:
In this protocol, we describe the specific steps required to prepare human postmortem brain samples for ultrastructural microglial analysis. A detailed procedure is provided to improve the ultrastructural quality of the samples, using aldehyde fixatives followed by immunoperoxidase staining of allograft inflammatory factor 1 (AIF1, also known as IBA1), a marker of myeloid cells, and cluster of differentiation 68 (CD68), a marker of phagolysosomal activity. Additionally, we describe an osmium-thiocarbohydrazide-osmium (OTO) post-fixation method that preserves and increases the contrast of cellular membranes in human postmortem brain samples, as well as the steps necessary to acquire scanning electron microscopy (SEM) images of microglial cell bodies. In the last section, we cover the quantitative analysis of various microglial cytoplasmic organelles and their interactions with other parenchymal elements.
Insights
This protocol details preparing human postmortem brain samples for ultrastructural analysis of microglia. It enhances sample quality for examining microglial cells and their functions using advanced microscopy techniques.
Area of Science:
- Neuroscience
- Cell Biology
- Microscopy
Background:
- Microglial cells are crucial for brain health and disease.
- Understanding microglial ultrastructure is vital for neurological research.
- Existing methods for postmortem brain tissue analysis have limitations.
Purpose of the Study:
- To provide a detailed protocol for preparing human postmortem brain samples for ultrastructural microglial analysis.
- To optimize sample quality for high-resolution imaging of microglia.
- To enable detailed examination of microglial morphology and function.
Main Methods:
- Aldehyde fixation and immunoperoxidase staining for myeloid cell marker (AIF1/IBA1) and phagolysosomal activity marker (CD68).
- Osmium-thiocarbohydrazide-osmium (OTO) post-fixation for enhanced membrane contrast.
- Scanning electron microscopy (SEM) for imaging microglial cell bodies.
Main Results:
- The protocol yields high-quality ultrastructural preservation of human postmortem brain samples.
- Immunostaining effectively labels microglial cells and their phagolysosomal activity.
- OTO fixation significantly improves cellular membrane visualization.
- SEM imaging allows detailed analysis of microglial cytoplasmic organelles and interactions.
Conclusions:
- This protocol offers a robust method for ultrastructural microglial analysis in human postmortem brain tissue.
- The described techniques facilitate detailed investigation of microglial morphology, organelle function, and cellular interactions.
- This approach will advance research into neurological disorders by providing deeper insights into microglial behavior.
