Cell Biology JoVE (Open Access) Citable · DOI

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

Name: Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture
Uploaded: 2026-06-17
Duration: 10 min 38 s
Description: The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

DOI: 10.3791/60223-v

What you'll learn

✓ Establish and culture mouse prostate organoids from sorted epithelial cells
✓ Extract protein lysate from organoids for Western blot analysis
✓ Perform whole-mount immunofluorescence and confocal microscopy on prostate organoids

Protocol

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

Difficulty

advanced

Total time

~3–5 days (organoid establishment to imaging analysis)

Model organism

Mouse (prostate epithelial cells)

Biosafety

BSL-1

Steps

Plate sorted prostate epithelial cells into organoid culture

Seed isolated and sorted mouse prostate epithelial cells into primary 3D organoid culture conditions to establish self-renewing epithelial structures.

▶ 00:52

Extract protein lysate from prostate organoids

Harvest and lyse prostate organoids to prepare protein samples suitable for Western blot analysis of lineage marker expression.

▶ 03:29

Fix and stain organoids for immunohistochemical analysis

Chemically fix prostate organoids and apply immunofluorescent antibody staining to visualize specific lineage markers and cellular differentiation.

▶ 04:50

Clear tissue and mount stained organoids for imaging

Optically clear fixed organoids and mount them on slides to prepare specimens for high-resolution whole-mount confocal microscopy.

▶ 06:44

Analyze lineage marker expression by dual imaging methods

Evaluate differentiation capacity by comparing protein expression patterns detected via Western blot and confocal microscopy visualization in prostate organoids.

▶ 07:57

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