Home › Immunology › Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart
Immunology JoVE (Open Access) Citable · DOI

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

DOI: 10.3791/52644-v
What you'll learn
  • ✓ Prepare embryonic mouse hearts for immunofluorescence analysis via snap-freezing and cryoprotection
  • ✓ Perform immunofluorescence staining to visualize cardiomyocyte-specific structures
  • ✓ Acquire and analyze confocal images of cardiac development in mouse embryos
Protocol

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

Difficulty
advanced
Total time
~3-4 days (including tissue collection, fixation, cryoprotection, sectioning, staining, and imaging)
Model organism
Mouse (embryonic)
Biosafety
BSL-1

Steps

1
Snap-freeze embryonic mouse hearts

Rapidly freeze dissected embryonic mouse hearts in liquid nitrogen to preserve cellular and tissue morphology for subsequent processing.

▶ 01:03
2
Fix, cryoprotect, and embed heart tissue

Treat snap-frozen hearts with paraformaldehyde fixation, cryoprotect with sucrose gradients, and embed in OCT compound for cryosectioning.

▶ 02:02
3
Cryosection embedded heart tissue

Cut thin serial sections from OCT-embedded hearts using a cryostat to generate slides for immunofluorescence staining.

▶ 02:55
4
Perform immunofluorescence staining

Apply primary and secondary antibodies to tissue sections to label cardiomyocyte-specific structures with fluorescent markers.

▶ 03:51
5
Acquire and analyze confocal images

Image stained sections using confocal microscopy and analyze acquired images to assess cardiomyocyte morphology and development.

▶ 05:16
6
Interpret cardiomyocyte developmental results

Review representative confocal images and image analysis results demonstrating changes in cardiomyocyte structure during embryonic heart development.

▶ 08:30
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