The Use of Immunofluorescent Array Tomography to Study the Three-Dimensional Microstructure of Murine Blood Vessels

Abstract

The purpose of this study was to develop the methods needed to enable the application of Immunofluorescent Array Tomography (IAT), a novel three-dimensional (3D) microscopy technique, to murine blood vessels. The anterior and posterior regions of the infrarenal aorta of 8-10 week old C57BL6 mice were evaluated. Staining and image analysis methods were developed. Antibody selection, primary antibody concentration, co-staining with multiple primary antibodies, and the multi-cycle staining design were optimized to produce positive and specific staining of elastin (1:50 dilution), smooth muscle cell actin (SMCA, 20 lg mL À1 ), and collagen type I (10 lg mL À1 ). Non-specific interactions were limited by optimizing secondary antibody staining conditions (1:200 dilution, 30 min). SMCA and collagen type I were stained in the first cycle, elastin was stained in the second cycle, and nuclei were stained in both cycles. Algorithms were developed to quantify volume fractions of medial elastin, SMCA, and nuclei, as well as adventitial collagen type I. Elastin thickness, spacing between elastin lamellae, elastin fragmentation, media wall thickness, nuclei aspect ratio, and nuclei amount were also quantified. We have qualitatively and quantitatively characterized the 3D microstructure and cellular morphology of the anterior and posterior infrarenal murine aorta using IAT.

MicroRNA miR-29b regulates diabetic aortic remodeling and stiffening

“…Elastin fragmentation was quantified in the histological images using elastin fluorescence morphometric analysis (ImageJ). As previously described, 32 each continuous set of pixels that formed a connected group was defined as an object. An elastin fragmentation index was defined as the ratio of the number of elastin objects to the area of elastin objects.…”

Three-Dimensional Microstructural Changes in Murine Abdominal Aortic Aneurysms Quantified Using Immunofluorescent Array Tomography

“…Ribbons were washed several times with PBS. The secondary antibodies, donkey anti-rabbit Alexa Fluor 488 (isotype: IgG; manufacturer’s concentration: 2 mg/ml; catalogue number: A-21206; lot number: 556439; Invitrogen, Carlsbad, CA) and donkey anti-goat Alexa Fluor 647 (isotype: IgG; manufacturer’s concentration: 2 mg/ml; catalogue number: A-21447; lot number: 552224; Invitrogen), were applied directly to the ribbons at 10 µg/ml in blocking solution for 30 min at room temperature (Saatchi et al 2011). Ribbons were washed several times with PBS, then with distilled water.…”

“…Blocking solution consisted of 0.05% Tween (Tween 20; Acros Organics, Morris Plains, NJ) and 0.1% bovine serum albumin (BSA Fraction V; Fisher Scientific, Pittsburgh, PA) in Tris buffer. In the first staining cycle, actin alpha 2 smooth muscle primary antibody (host species: rabbit; isotype: IgG; manufacturer's concentration: 200 µg/ml; catalogue number: NB600-531; lot number: 100105; Novus Biologicals, Littleton, CO) at 20 µg/ml and collagen type I primary antibody (Col1A1 [D-13]; host species: goat; isotype: IgG; manufacturer's concentration: 200 µg/ ml; catalogue number: SC-25974; lot number: F2609; Santa Cruz Biotechnology, Santa Cruz, CA) at 10 µg/ml in the described blocking solution were applied directly onto the ribbons, overnight at 4C (Saatchi et al 2011). Ribbons were washed several times with PBS.…”

“…Three-dimensional qualitative renderings of the 63× image sets were created using AxioVision software (AxioVision 4.7; Zeiss) for both the anterior and posterior vessel wall regions. Custom algorithms using MATLAB (MATLAB R2007b; MathWorks, Natick, MA) were developed for quantitative analysis of microstructure and cellular morphology (Saatchi et al 2011). Aligned segmented gray-scale image series were used for individual tissue constituent analysis, specifically elastin, SMCA, collagen type I, and nuclei.…”

“…With IAT, arrays of ultrathin serial sections are generated that undergo numerous cycles of antibody staining and imaging to identify multiple targets of interest in a single sample using immunofluorescence microscopy. IAT is superior to existing imaging techniques in terms of spatial resolution, quantitative analysis compatibility, specificity, and multiplexing capabilities (Micheva and Smith 2007; Saatchi et al 2011). Furthermore, 3D visualization of tissue and cellular ultrastructure using IAT will provide a unique perspective on aneurysm development for the first time.…”