Study of Temporal changes in microvessel leakiness during wound healing by Fluorescence recovery after photobleaching (FRAP) (CAT#: STEM-MT-0055-WXH)

Introduction

Wound healing is a complex and dynamic biological process that proceeds through three distinct and temporally overlapping phases: inflammation, proliferation and remodelling. Paramount to successful healing is wound closure, and the replacing of injured or necrotic tissue with granulation tissue. Formation of granulation tissue is, in turn, dependent on angiogenesis.
A time-resolved quantitative analysis of plasma flux across the vessel wall in BEVs is essential to the understanding of how a functional vascular plexus regenerates after injury. BEVs are the proximally perfused segments of newly formed angiogenic sprouts and their temporal development in relation to wound closure regulates the initial patterning of the regenerating vascular plexus.

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Principle Applications Procedure Materials Notes Related Products

Principle

Fluorescence recovery after photobleaching (FRAP) is a microscopy technique capable of quantifying the mobility of molecules within cells. By exploiting the phenomenon of photobleaching, fluorescent mole- cules within a region of interest can be selectively and irreversibly 'turned off'. It is capable of quantifying the two-dimensional lateral diffusion of a molecularly thin film containing fluorescently labeled probes, or to examine single cells.

Applications

• Characterization of the mobility of individual lipid molecules within a cell membrane.
• Analysis of molecule diffusion within the cell
• Study of protein interaction partners, organelle continuity and protein trafficking.

Procedure

1. An initial fluorescence of fluorescent molecules is measured in the region of interest (ROI).
2. The fluorescent molecules are rapidly photobleached by focusing the high-intensity laser beam onto the defined area.
3. The exchange of bleached molecules with unbleached molecules from the surrounding region is followed over time using a low-intensity laser.

Materials

• Optical microscope.
• Light source.
• Fluorescent probe.

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