Immunology JoVE (Open Access) Citable · DOI

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Name: Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia
Uploaded: 2026-06-17
Duration: 12 min 42 s
Description: Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

DOI: 10.3791/52031-v

What you'll learn

✓ Perform stereological quantification of leukocytes in ischemic brain tissue using optical fractionator method.
✓ Prepare single-cell brain suspensions and analyze leukocyte subsets by flow cytometry.
✓ Distinguish myeloid cell phenotypes in transient and permanent cerebral ischemia models.

Protocol

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Difficulty

advanced

Total time

~4-6 hours per animal (including ischemia induction, tissue processing, and analysis)

Model organism

Mouse

Biosafety

BSL-1

Steps

Occlude common carotid and middle cerebral arteries

Perform surgical ligation of the common carotid artery and distal middle cerebral artery to induce transient or permanent cerebral ischemia in anesthetized mice.

▶ 02:20

Perfuse and section ischemic brain tissue

Perform cardiac perfusion with fixative and prepare serial coronal brain sections for downstream stereological and flow cytometric analyses.

▶ 03:56

Quantify infiltrated neutrophils by stereology

Apply the optical fractionator method to systematically count and characterize neutrophil populations within defined regions of ischemic brain tissue sections.

▶ 05:36

Prepare brain tissue single-cell suspension

Mechanically and enzymatically dissociate ischemic brain tissue to generate a single-cell suspension enriched for leukocytes.

▶ 07:23

Analyze leukocyte subsets by flow cytometry

Perform flow cytometric characterization of brain leukocyte populations from naïve, sham, and ischemic mice to identify and quantify distinct myeloid cell subpopulations.

▶ 09:41

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