Basic NeuroscienceA zinc fixative for 3D visualization of cerebral capillaries and pericytes

- CD31 immunohistochemical staining of zinc-fixated, thick (100 μm) brain tissue resulted in complete visualization of cerebral capillaries in mice.
- Fluorescent co-localization of CD31 with the NG2 pericyte marker verified the use of CD31 as a pericyte marker for light microscopic stereological investigations.
- The general morphology of zinc-fixated tissue is comparable to aldehyde-based fixatives.
- The tissue shrinkage to 30% of the original volume is within an expected normal range.

BackgroundA large volume of data indicates that disturbances in the morphology and function of the capillary wall may play a causal role in several types of neurodegenerative disorders. We present a highly reproducible staining method for investigating the cerebral capillary network and the pericyte cells within the basement membrane in mice - a specie specific challenging task when uniform staining in thick sections was needed for confocal microscopy or a quantitative analysis, e.g. stereological investigation using 3D probes.New methodWe perfused C57BL6/Jbom mice and immersion fixated the brains with an aldehyde free zinc fixative, which is normally used for paraffin embedded tissues, and stained for CD31 and Collagen Type IV positive capillaries in 100 μm thick sections.ResultsUsing the milder zinc fixative allowed complete immunohistochemical visualization of the cerebral capillary network in 100 μm thick sections using CD31 or Collagen Type IV antibodies. Moreover CD31 or Collagen Type IV staining revealed the presence of pericytes, which was confirmed by a fluorescent co-localization with the NG2 pericyte marker.Comparison with existing methodsCompared with conventional aldehyde-based fixative, this method resulted in a homogeneous staining through the entire depth of thick sections with very limited background staining and well-preserved morphology.ConclusionsThis method is suitable for 3D stereological analysis of capillary networks and pericytes within thick brain sections using CD31 or Collagen Type IV antibodies.