Calculated levels of bound antibody correlated well (R2 = 0.52) with histologically determined numbers of infiltrating macrophages (Figure 5C and 5D), confirming that imaging with specific tracers can be indicative of local disease activity. for quantitative three-dimensional tissue assessments have been developed6. This has been accomplished through the development of fluorescent probes that emit light in the near-infrared (NIR) spectrum, offering low absorption, sensitive detectors, and monochromatic light sources7. While traditional cross-sectioning imaging techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), or ultrasound (US), rely mostly on physical parameters and visualize morphology, optical imaging can provide additional information on underlying molecular processes using endogenous or exogenous fluorescent probes8. Advances in molecular biology have helped to facilitate the generation of smart and targeted fluorescent molecular probes for an increasing number of targets. For example, receptor-mediated uptake and distribution in a given target area can be visualized using carbocyanine derivative-labeled antibodies9. The abundance of available antibodies, which can be labeled to function as specific tracers in otherwise inaccessible areas of the body, provides unprecedented insights Tiagabine hydrochloride into molecular and cellular processes in models of tumorigenesis and neurodegenerative, cardiovascular, immunologic, and inflammatory diseases7. In this study, we describe the use of fluorescence-mediated tomography in a murine model of colitis. Dextran sodium sulfate (DSS)-induced colitis is a standard chemically induced mouse model of intestinal inflammation that resembles inflammatory bowel disease (IBD)10. It Tiagabine hydrochloride is particularly useful to assess the contribution of the innate Serpinf1 immune system to the development of gut inflammation11. Since the recruitment, activation, and infiltration of monocytes and macrophages represent crucial steps in the pathogenesis of IBD, Tiagabine hydrochloride visualization of their recruitment and the kinetics of infiltration are essential to monitoring, for example, the effect of potential therapeutic substances in a preclinical setting12. We describe the induction of DSS colitis and demonstrate the tomography-mediated characterization of macrophage infiltration into the gut mucosa using fluorescence molecular tomography for the specific visualization of the monocyte/macrophage marker F4/8013. Additionally, we illustrate auxiliary and supplemental procedures, such as antibody labelling; the experimental setup; and analysis and interpretation of the obtained images, in correlation with conventional readouts such as disease activity indices, flow cytometry and histological analysis, and immunohistochemistry. We discuss limitations of this technique and comparisons to other imaging modalities. Protocol All animal experiments were approved by the Landesamt fr Natur, Umwelt und Verbraucherschutz (LANUV) Nordrhein-Westfalen according to the German Animal Protection Law (Tierschutzgesetz). 1. Materials and Experimental Setup Animal care. Use sex- and age-matched mice of any DSS-susceptible strain (C57BL/6) at 20-25 g bodyweight. Plan at least five or more mice per experimental group and house the mice according to local animal care guidelines. Provide a standard rodent chow diet and autoclaved drinking water application. Determine the final antibody concentration and labeling ratio by spectrophotometry. Measure the protein concentration at an absorption of 250-330 nm and consider additional absorption by the dye. Correct the maximum absorption at 280 nm (protein) by 11% of the maximum absorption at 750 nm (next step) for Cyanine7 labeling. Measure a dilution of the compound (typically 1:10) at 250-800 nm and extract the concentration of Cyanine7 at 750 nm. Determine the dye-to-protein ratio as: dye/antibody = maximum absorption at 750 nm / 200,000 / (maximum absorption at 280 nm – 0.11 x max absorption at 750 nm) / 170,000. Keep the antibody solution at 4 C and shield it from light to avoid bleaching before injection. Load the necessary antibody solution volume in a sterile syringe immediately before injection and shield from light until it is used. Determine the optimal timing of probe injection and the scanning procedure, depending on tracer pharmacokinetics. Anesthetize the mice using 1.5 – 2.5% inhaled isoflurane in oxygen or place them securely in a dedicated restrainer for the tail vein injection of the labeled antibodies . For full-length antibodies,.
Calculated levels of bound antibody correlated well (R2 = 0
