Home › Cell Biology › High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis
Cell Biology JoVE (Open Access) Citable · DOI

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

DOI: 10.3791/56407-v
What you'll learn
  • ✓ Prepare brain free-floating sections for high-resolution confocal imaging.
  • ✓ Perform immunofluorescence staining to label BBB cells and organelles.
  • ✓ Acquire and process confocal images for 3D reconstruction of the neurovascular unit.
  • ✓ Quantify transcytotic vesicles and intracellular structures at the blood-brain barrier.
Protocol

Biopharma Insights Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

Difficulty
advanced
Total time
~3–4 days (sectioning, staining, imaging, and image processing per brain)
Model organism
Mouse
Biosafety
BSL-1

Steps

1
Generate brain free-floating sections

Prepare mouse brain tissue and generate thin free-floating cryosections suitable for immunofluorescence and confocal imaging of the blood-brain barrier.

▶ 00:53
2
Label cells and organelles by immunofluorescence

Apply primary and secondary antibodies to stain endothelial cells, pericytes, and intracellular organelles (vesicles, mitochondria) within the neurovascular unit.

▶ 02:19
3
Acquire high-resolution confocal images

Use confocal microscopy to capture high-resolution optical sections of labeled blood-brain barrier structures with appropriate pinhole and laser settings.

▶ 04:27
4
Process images and perform 3D reconstruction

Apply image processing algorithms to denoise, register, and segment confocal stacks, then construct three-dimensional models of capillaries and vesicles.

▶ 06:21
5
Quantify transcytotic vesicles and structures

Extract volumetric and morphological data from reconstructed 3D models to measure transcytosis and characterize intracellular organelle distribution at the BBB.

▶ 09:04
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