Double Immunofluorescence Staining for Blood Vessel Study

Detection of blood vessels and assessment of their functional state is an acute issue of modern histology.

The aim of the study was to develop a double immunofluorescence staining technique to study different types of blood vessels in the rat lung.

Material and methods. The study included samples of the right and left lungs of sexually mature (3–5 months) male Wistar rats (n=5). Rabbit polyclonal antibodies to von Willebrand factor (Agilent, USA) and mouse monoclonal (clone 1A4) antibodies to smooth muscle α-actin (Agilent, USA) were applied to detect blood vessels. Sections were analyzed using the method of confocal laser microscopy.

Results. In the study the authors designed a double immunofluorescence protocol, which allows highly selective detection of blood vessels of various types in the rat lung. The use of von Willebrand factor as one of the markers facilitates blood vessel endothelium identification. Application of the second marker – smooth muscle α-actin – allows identifying a layer of smooth myocytes; and their structural features provide detection of the blood vessel type (whether it belongs to the arterial or venous bed). The proposed protocol is characterized by good reproducibility and allows producing high quality drugs. Due to the high specific immunoreactivity of the antibodies used, the stage of thermal unmasking of antigens can be removed from the staining protocol, which makes it less time-consuming, shortens the preparation time of the sections and ensures their better preservation. The use of von Willebrand factor and smooth muscle α-actin as markers allow evaluating not only the structure, but also the functional status of the studied blood vessels. This provides application of the proposed method for scientific and
clinical diagnostic studies.

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