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The present protocol describes light-sheet fluorescent microscopy and automated software-assisted methods to visualize and precisely quantify proliferating and dormant Trypanosoma cruzi parasites and T cells in intact, cleared organs and tissues. These techniques provide a reliable way to evaluate treatment outcomes and offer new insights into parasite-host interactions.
Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi (T. cruzi), is an obligate intracellular parasite with a diverse biology that infects several mammalian species, including humans, causing cardiac and digestive pathologies. Reliable detection of T. cruzi in vivo infections has long been needed to understand Chagas disease's complex biology and accurately evaluate the outcome of treatment regimens. The current protocol demonstrates an integrated pipeline for automated quantification of T. cruzi-infected cells in 3D-reconstructed, cleared organs. Light-sheet fluorescent microscopy allows for accurately visualizing and quantifying of actively proliferating and dormant T. cruzi parasites and immune effector cells in whole organs or tissues. Also, the CUBIC-HistoVision pipeline to obtain uniform labeling of cleared organs with antibodies and nuclear stains was successfully adopted. Tissue clearing coupled with 3D immunostaining provides an unbiased approach to comprehensively evaluate drug treatment protocols, improve the understanding of the cellular organization of T. cruzi-infected tissues, and is expected to advance discoveries related to anti-T. cruzi immune responses, tissue damage, and repair in Chagas disease.
Chagas disease, caused by the protozoan parasite T. cruzi, is among the world's most neglected tropical diseases, causing approximately 13,000 annual deaths. The infection often progresses from an acute to a chronic stage producing cardiac pathology in 30% of the patients, including arrhythmias, heart failure, and sudden death1,2. Despite the strong host immune response elicited against the parasite during the acute phase, low numbers of parasites chronically persist throughout the host's life in tissues such as the heart and skeletal muscle. Several factors, including the delayed onset of ada....
This study was carried out in strict accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and Association for Assessment and Accreditation of Laboratory Animal Care accreditation guidelines. The Animal Use Protocol (control of T. cruzi infection in mice-A2021 04-011-Y1-A0) was approved by the University of Georgia Institutional Animal Care and Use Committee. B6.C+A2:A44g-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J.......
CUBIC fixed tissues were washed with PBS to remove fixatives and then incubated with CUBIC-L cocktails, a basic buffered solution of amino alcohols that extract pigments and lipids from the tissue resulting in decolorization of tissue while maintaining tissue architecture. Grid lines in the paper can be seen through the tissues indicating an appropriate clearing of the organs (Figure 2A). After delipidation, tissues were washed and immersed in CUBIC-R+ and mounting solution .......
The absence of extensive, whole-organ imaging of parasites and the immune response limits the understanding of the complexity of the host-parasite interactions and impedes the evaluation of therapies for Chagas disease. The present study adopted the CUBIC pipeline to clarify and stain intact organs and tissues of T. cruzi-infected mice.
Multiple tissue clearing protocols were tested in this study (PACT32, ECi33, FLASH34.......
We thank Dr. Etsuo Susaki for their valuable help and recommendations regarding tissue-clearing and immunostaining protocols. Also, we are grateful to M. Kandasamy from the CTEGD Biomedical Microscopy Core for technical support using LSFM and confocal imaging. We also thank all the members of Tarleton Research Group for helpful suggestions throughout this study.
....Name | Company | Catalog Number | Comments |
1-methylimidazole | Millipore Sigma | 616-47-7 | |
2,3-Dimethyl-1-phenyl-5-pyrazolone (Antipyrine | TCI | D1876 | |
6-wells cell culture plates | ThermoFisher Scientific | 140675 | |
AlexaFluor 647 anti-mouse Fab fragment | Jackson Immuno Research Laboratories | 315-607-003 | |
AlexaFluor 647 anti-rabbit Fab fragment | Jackson Immuno Research Laboratories | 111-607-003 | |
anti-GFP nanobody Alexa Fluor 647 | Chromotek | gb2AF647-50 | |
anti-RFP | Rockland | 600-401-379 | |
anti-α-SMA | Sigma | A5228 | |
B6.C+A2:A44g-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mouse | The Jackson Laboratory | Strain #007914 | Common Name: Ai14 , Ai14D or Ai14(RCL-tdT)-D |
B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze/J mouse | The Jackson Laboratory | Strain #007914 | Common Name: Ai14 , Ai14D or Ai14(RCL-tdT)-D |
BOBO-1 Iodide | ThermoFisher Scientific | B3582 | |
Bovine serum albumin (BSA) | Sigma | #A7906 | |
C57BL/6J-Tg(Cd8a*-cre)B8Asin/J mouse | The Jackson Laboratory | Strain #032080 | Common Name: Cd8a-Cre (E8III-Cre) |
CAPSO | Sigma | #C2278 | |
Cleaning wipes Kimwipes | Kimberly-Clark | T8788 | |
Confocal Laser Scanning Microscope | Zeiss | LSM 790 | |
CUBIC-HV 1 3D immunostaining kit | TCI | C3699 | |
CUBIC-HV 1 3D nuclear staining kit | TCI | C3698 | |
CUBIC-L | TCI | T3740 | |
CUBIC-P | TCI | T3782 | |
CUBIC-R+ | TCI | T3741 | |
Cyanoacrylate-based gel superglue | Scotch | 571605 | |
DiR (DiIC18(7); 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) Company: Biotium | Biotium | #60017 | |
Ethylene diamine tetra acetic acid (EDTA) | Millipore Sigma | 60-00-4 | |
Falcon Centrifuge tubes 15 mL | Corning | CLS430791 | |
Falcon Centrifuge tubes 50 mL | Corning | CLS430290 | |
Formalin | Sigma-Aldrich | HT501128 | |
Heparin | ThermoFisher Scientific | J16920.BBR | |
Hyaluronidase | Sigma | #H3884 or #H4272 | |
Imaris File Converter x64 | BitPlane | v9.2.0 | |
Imaris software | BitPlane | v9.3 | |
ImSpector software | LaVision BioTec, Miltenyi Biotec | v6.7 | |
Intravenous injection needle 23-G | Sartori, Minisart Syringe filter | 16534 | |
Kimwipes | lint free wipes | ||
Light-sheet fluorescent microscope | Miltenyi Biotec | ULtramicroscope II imaging system | |
Methanol | ThermoFisher Scientific | 041838.K2 | |
Micropipette tips, 10 µL, 200 µL and 1,000 µL | Axygen | T-300, T-200-Y and T-1000-B | |
Motorized pipet dispenser | Fisher Scientific, Fisherbrand | 03-692-172 | |
Mounting Solution | TCI | M3294 | |
N-butyldiethanolamine | TCI | B0725 | |
Nicotinamide | TCI | N0078 | |
N-Methylnicotinamide | TCI | M0374 | |
Paraformaldehyde (PFA) | Sigma-Aldrich | 158127 | |
Phosphate buffered saline (PBS) | Thermo Fisher Scientific | 14190-094 | |
RedDot 2 Far-Red Nuclear Stain | Biotium | #40061 | |
Sacrifice Perfusion System | Leica | 10030-380 | |
Scissors | Fine Science Tools | 91460-11 | |
Serological pipettes | Costar Sterile | 4488 | |
Shaking incubator | TAITEC | BR-43FM MR | |
Sodium azide (NaN3) | ThermoFisher Scientific | 447815000 | |
Sodium carbonate (Na2CO3) | ThermoFisher Scientific | L13098.36 | |
Sodium Chloride (NaCl) | ThermoFisher Scientific | 447302500 | |
Sodium hydrogen carbonate (NaHCO3) | ThermoFisher Scientific | 014707.A9 | |
SYTOX-G Green Nucleic Acid Stain | ThermoFisher Scientific | S7020 | |
Triton X-100 | Sigma-Aldrich | T8787 |
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