Home › Cell Biology › Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy
Cell Biology JoVE (Open Access) Citable · DOI

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

DOI: 10.3791/51388-v
What you'll learn
  • ✓ Perform intravital immunofluorescence staining on live tissue using in situ labeling
  • ✓ Acquire high-resolution matrix dynamics using epifluorescence and two-photon microscopy
  • ✓ Visualize fibrillar and mesh-like extracellular matrix remodeling in real time
  • ✓ Apply long-term imaging protocols to wound healing and tumor microenvironment studies
Protocol

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

Difficulty
advanced
Total time
~3-4 hours per mouse (including tumor cell preparation, ear surgery, staining, and imaging)
Model organism
Mouse (tumor xenograft model in ear)
Biosafety
BSL-1

Steps

1
Prepare tumor cells and perform ear injection

Culture and harvest tumor cells for injection. Prepare mouse ear tissue for cell implantation and perform intradermal or subcutaneous tumor cell injection into the ear.

▶ 01:50
2
Perform surgical preparation of mouse ear

Anesthetize mouse and expose the ear tissue. Clear hair, prepare the surgical field, and position the ear for imaging access using stereomicroscopy.

▶ 03:36
3
Conduct immunofluorescence staining during intravital imaging

Apply fluorescently-labeled antibodies or fluorescent probes directly to live tissue in situ. Perform staining while imaging with stereomicroscope to visualize matrix component binding.

▶ 04:40
4
Acquire intravital immunofluorescence images

Capture high-resolution time-lapse images of extracellular matrix dynamics using epifluorescence and/or two-photon microscopy. Document fibrillar and mesh-like matrix remodeling over extended imaging sessions.

▶ 07:06
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