Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

Summary

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.

Abstract

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.

Introduction

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 death^1,2. 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 adaptive immune responses and the presence of non-replicating forms of the parasite, may contribute to the capacity of T. cruzi to avoid a complete elimination by the immune system^3,4,5,6. Furthermore, non-replicating dormant forms of the parasite display a low susceptibility to trypanocidal drugs and may in part be responsible for the treatment failure observed in many cases^7,8.

The development of new imaging techniques provides an opportunity to gain insight into the spatial distribution of the parasites in the infected tissues and their relationship with the immune cells involved in their control. These characteristics are crucial for a better understanding of the processes of parasite control by the immune system and tracking the rare dormant parasites present in chronic tissues.

Light-sheet fluorescence microscopy (LSFM) is one of the most comprehensive and unbiased methods for 3D imaging of large tissues or organs without thin-sectioning. Light-sheet microscopes utilize a thin sheet of light to only excite the fluorophores in the focal plane, reduce photobleaching and phototoxicity of samples, and record images of thousands of tissue layers using ultra-fast cameras. The high level of tissue transparency necessary for the proper penetration of the laser light in tissues is obtained by homogenizing the refractive index (RI) following tissue delipidation and decolorization, which reduces the scattering of light and renders high-quality images^9,10,11.

Tissue clearing approaches have been developed for the imaging of whole mice^12,13,14, organoids^15,16,17, organs expressing reporter fluorescent markers^{18,19,20,21,22,23}, and recently a limited number of human tissues²⁴. The current methods for tissue clearing are classified into three families: (1) organic solvent-based methods such as DISCO protocols^25,26, (2) hydrogel-based methods, such as CLARITY²⁷, and aqueous methods, such as CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis)^18,19,28,29. CUBIC protocols maintain organ shape and tissue integrity, preserving the fluorescence of endogenously expressed reporter proteins. The most recent update of this technique, CUBIC-HistoVision (CUBIC-HV), also permits the detection of epitopes using fluorescently-tagged antibodies and DNA labeling²⁸.

In the present protocol, the CUBIC pipeline for detecting T. cruzi expressing fluorescent proteins in clarified intact mouse tissues was used. Optically transparent tissues were LSFM imaged, 3D reconstructed, and the precise total number of T. cruzi infected cells, dormant amastigotes, and T cells per organ were automatically quantified. Also, this protocol was successfully adopted to obtain uniform labeling of cleared organs with antibodies and nuclear stains. These approaches are essential for understanding the expansion and control of T. cruzi in infected hosts and are useful for fully evaluating chemo- and immuno-therapeutics for Chagas disease.

Protocol

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)26Sor^{tm14(CAG-tdTomato)Hze}/J, B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J and C57BL/6J-Tg(Cd8a*-cre)B8Asin/J mice (female, 1-2 months old) were used for the present study. The mice were obtained from commercial sources (see Table of Materials).

1. Infection, perfusion, and dissection

1. Intraperitoneally infect mice with tissue culture-derived trypomastigotes of Colombiana (DTU TcI) or Brazil (DTU TcV) T. cruzi strain expressing tdTomato or GFP fluorescent proteins, respectively. The infection dose could range from 50,000 to 200,000 trypomastigotes diluted in 100 µL of 1x Phosphate-Buffered Saline (PBS).
   NOTE: Specific details about the generation of reporter parasites and mouse models of infection are available in Canavaci et al.²⁹ and Bustamante et al.³⁰.
2. Euthanize the mice by carbon dioxide inhalation at a flow rate of 3-7 L/min. As soon as the animals stop showing any pedal reflex, make a longitudinal incision through the skin from the abdomen towards the sternum. Then cut the body wall from the abdomen and continue through the ribs on each side of the thorax until the sternum can be lifted away, exposing the heart.
3. As described in Figure 1, make a 2.5 mm incision in the heart's right auricle and collect the draining blood using a 1 mL micropipette tip.
4. Insert a butterfly needle (connected to one end of the perfusion system) into the apex of the left ventricle until it reaches the ascending aorta. Use gel-based glue (see Table of Materials) to seal the inlet hole around the needle and maintain the needle in position during perfusions.
5. Perfuse the mice³⁰ with 50 mL of cold Heparin-PBS (pH 7.4, 10 U/mL of Heparin) or until the fluid that comes out of the mouse toward the collection tray is clear of blood.
6. Perfuse with 50 mL of cold 4% (w/v) paraformaldehyde (PFA) (pH 7.4) in PBS.
   NOTE: PFA tissue fixation is likely one of the critical steps of the protocol, especially for maintaining epitope structures and subsequent immunodetection. PFA degrades over time, so it must be freshly prepared. The pH of the solution is also important to avoid over-fixation, which could lead to poor clearing of tissues.
   CAUTION: PFA is moderately toxic by skin contact. Acute exposure is also highly irritating to the nose, eyes, and throat. Long-term exposure to low levels in the air or skin may cause skin irritation such as dermatitis and itching, and asthma-like respiratory problems. Wear face and eye protection and do not breathe dust, gas, mist, fumes, or vapors.
   1. Following the PFA perfusion, perfuse the mouse with 100 mL of CUBIC-P buffer to reach the clarification levels mentioned in step 1.5.
   2. To prepare CUBIC-P buffer, dissolve 10% N-butyldiethanolamine, 5% Triton X-100, and 5% 1-N-Methylimidazole in double-distilled water or use the commercial cocktails (see Table of Materials).
      NOTE: Steps 1.6.1.-1.6.2. are recommended only for highly pigmented organs such as the heart and kidney since they require a pre-clearing step to obtain increased levels of transparency. After using CUBIC-P, dissect the organs and proceed directly to step 2: Tissue clearing.
7. Dissect the tissue samples/organs³⁰ to be imaged and post-fix them in 4% (w/v) PFA in PBS (~10 mL/whole organ) overnight (ON) at 4 °C with gentle shaking (no more than 5 x g) in 50 mL conical tubes.
   NOTE: All the incubations from hereafter must be performed on tubes laying horizontally at 20-30 °C protected from light.
8. Wash the sample in 10 mL of PBS (supplemented with 0.05% sodium azide (NaN₃) for 3 h (three times) at room temperature (RT) with gentle shaking (5 x g).
   ​NOTE: Tissues can be frozen by incubating in 10 mL of 30% sucrose in PBS with gentle shaking (5 x g) at 4 °C ON in 50 mL conical tubes. After tissues sink to the bottom of the tube, embed them in the OCT compound and keep them at -80 °C. Thaw at RT until the OCT compound completely melts, then wash in PBS (~10 mL/whole organ) ON at 4 °C with gentle shaking (5 x g) in 50 mL conical tubes. Proceed to step 2.

2. Tissue clearing

NOTE: All the tissue clearings performed in this work were done using CUBIC protocol I²². Three different cocktails were used: CUBIC-P for delipidation and rapid decolorization during perfusions, CUBIC-L for delipidation and decolorization, and CUBIC-R for RI matching. DNA staining and immunostainings were performed using CUBIC-HV 1 3D nuclear staining kit and CUBIC-HV 1 3D immunostaining kit, respectively (see Table of Materials).

1. Immerse individual organs in 10 mL of 50% water-diluted CUBIC-L (see Table of Materials) with gentle shaking (5 x g) at RT (ON) in 50 mL conical tubes. Keep tubes flat on the shaking plate.
   NOTE: To avoid tissue damage, organs are maintained in the same tube, and solutions are collected using a vacuum system. To prepare CUBIC-L, dissolve 10% N-butyldiethanolamine and 10% Triton X-100 in double-distilled water using the commercial cocktails (see Table of Materials).
   CAUTION: CUBIC-L causes serious eye damage.Wear eye and face protection. Dispose to an approved waste disposal plant.
2. Immerse sample in 10 mL of 100% CUBIC-L for 6 days (refreshing the solution on day 3).
   NOTE: At the end of this incubation period, the tissues must be almost completely transparent.
3. Wash the transparent organs with PBS (supplemented with 0.05% NaN₃) for 2 h (three times) at 37 °C with gentle shaking (5 x g). Transfer tissues to a new 50 mL conical tube with each wash to remove Triton X-100.
   NOTE: As described in Figure 2B, if the goal of the experiment is to visualize endogenously-expressed reporter proteins from transgenic T. cruzi parasites or mice (Figure 2C-F), skip steps 3, 4, and 5 and continue directly to step 6.

3. DNA staining

1. Dilute the commercially available nucleic acid dye (see Table of Materials) in 5 mL of staining buffer (included in the kit) at 1/2,500 dilution.
2. Immerse tissue in the nuclear dye solution and incubate at 37 °C with gentle rotation for 5 days using 15 mL conical tubes in a standing position.
3. Wash with 15 mL of 3D nuclear staining wash buffer (included in the kit) for 2 h (three times) at RT with gentle shaking (5 x g).
   ​NOTE: Other DNA dyes can be used in these concentrations and incubation times: DAPI (included in the kit): 1/200, 5 days incubation; BOBO-1: 1/400, 5 days incubation; Propidium Iodide (PI) (included in the kit): 1/100, 3 days incubation; RedDot2: 1/250, 3 days incubation. If the specific aim of the experiment is to visualize both endogenously-expressed reporter proteins and use nuclear stains, skip steps 4 and 5 and continue directly to step 6.

4. Extracellular matrix (ECM) digestion

NOTE: Hyaluronidase digestion of the ECM must be performed to facilitate the proper penetration of the antibodies into deep regions of the tissues²⁸.

1. Immerse individual organs in 15 mL of hyaluronidase reaction buffer for 2 h at 37 °C in a 50 mL conical tube in flat position protected from light.
   NOTE: To prepare hyaluronidase reaction buffer, dissolve 10 mM of CAPSO; 150 mM of Sodium Chloride (NaCl), and 0.05% of NaN₃ (see Table of Materials) in double-distilled water and adjust pH to 10.
2. Prepare Enzyme Solution by mixing 75 µL of 20 mg/mL of hyaluronidase stock into 425 µL of hyaluronidase reaction buffer. To prepare 20 mg/mL of hyaluronidase stock, dissolve hyaluronidase in 50 mM of Carbonate buffer, 150 mM of NaCl, 0.01% of BSA, and 0.05% of NaN₃ (see Table of Materials). Adjust pH to 10 and aliquot in volumes of 77 µL at -30 °C.
3. Discard hyaluronidase reaction buffer using a pipette and immerse the organ in the Enzyme Solution (500 µL in a 15 mL conical tube) in a standing position protected from light for 24 h at 37 °C with gentle shaking (5 x g).
4. Wash the sample in 15 mL of hyaluronidase wash buffer in a 50 mL conical tube in a horizontal position protected from light for 2 h (three times) at 37 °C with gentle shaking (5 x g).
   1. To prepare hyaluronidase wash buffer, dissolve 50 mM of Carbonate buffer, 150 mM of NaCl, 0.1% (v/v) of Triton X-100, 5% (v/v) of Methanol, and 0.05% NaN₃. Adjust pH to 10. To prepare 10x Carbonate buffer-NaCl stock, dissolve 2.96 g of Sodium Carbonate, 1.86 g of Sodium Hydrogen Carbonate, and 8.77 g of NaCl in 100 mL of double-distilled water with 0.05% NaN₃ (see Table of Materials) and adjust pH to 10.

5. Immunostaining

1. Label vasculature using anti-α-SMA (alpha-small muscle actin, see Table of Materials) antibodies following the steps below.
   1. Generate primary plus conjugated Fab fragment secondary antibody complex. Start this reaction 1.5 h prior to the staining procedure.
      1. Calculate the required amount of primary and secondary antibodies (mix at a 1:0.5 ratio by weight).
         NOTE: For the primary antibody anti-α-SMA, 3.5 µg is needed to label the entire heart or a fragment of skeletal muscle of similar dimensions. For 2.5 mg/mL product, 3.5/2.5 = 1.4 µL of antibody solution is needed. For secondary antibody AlexaFluor 647 anti-mouse Fab fragment, 1.75 µg is needed to label the entire heart or a fragment of skeletal muscle of similar dimensions. For 1.7 mg/mL product, 1.75/1.7 = 1 µL of antibody solution is needed.
      2. Mix primary and secondary antibodies in an amber 2 mL tube and incubate for 1.5 h at 37 °C.
   2. For buffer exchange, mix 7.5 mL of 2x HV1 3D immunostaining buffer (included in the kit, see Table of Materials) with 7.5 mL of double-distilled water and immerse the tissue sample for 1.5 h at 32 °C with gentle shaking (5 x g) in a 15 mL conical tube in a horizontal position. Start this reaction simultaneously as the generation of antibody complex (1.5 h prior to the immunostaining procedure).
2. Perform 3D immunostaining following the steps below.
   1. In a 15 mL conical tube, prepare the antibody staining solution following Supplementary File 1.
   2. Collect the tissue sample from the buffer exchange media (step 5.1.2) and immerse it in the antibody staining solution. Incubate tissues individually for 1 week at 32 °C with gentle shaking (5 x g) of the tubes in a standing position protected from light. Seal the tube with paraffin film to avoid evaporation.
   3. Move to 4 °C and incubate ON in a standing position.
   4. Cool 1x HV1 3D immunostaining wash buffer (included in the kit, see Table of Materials) to 4 °C and wash the sample with 15 mL buffer (two times) for 30 min each at 4 °C with gentle shaking (5 x g). Keep 15 mL conical tubes in a horizontal position until step 5.2.7.
   5. Dilute formalin to 1% in 1x HV1 3D immunostaining wash buffer and immerse the sample in 8 mL of the solution for 24 h at 4 °C with gentle shaking (5 x g).
   6. Incubate in fresh 1% formalin solution for 1 h at 37 °C with gentle shaking (5 x g).
   7. Wash in 15 mL of PBS for 2 h at 25 °C with gentle shaking (5 x g).
3. Boost tdTomato signal using anti-Red Fluorescent Protein (RFP) antibodies following the steps below.
   1. Follow the same incubation times and temperatures as in step 5.2.2. Calculate the amount of primary and secondary antibodies (see Table of Materials) and mix them at a 1:0.5 ratio by weight.
      NOTE: For primary antibody anti-RFP, 5 µg is needed to label the entire heart or a fragment of skeletal muscle of similar dimensions. For 1.25 mg/mL product, 5/1.25 = 4 µL of the solution is required. For secondary antibody Alexa Fluor 647 anti-rabbit Fab fragment, 2.5 µg is needed to label the entire heart or a fragment of skeletal muscle of similar dimensions. For 1.5 mg/mL product, 2.5/1.5 = 1.7 µL of the solution is needed.
   2. Prepare the antibody staining solution as mentioned in Supplementary File 1.
4. Boost GFP signals using anti-GFP nanobodies following the steps below.
   1. Follow the same incubation times and temperatures as mentioned in step 5.2.2.
      NOTE: Anti-GFP nanobodies (see Table of Materials) are conjugated with Alexa Fluor 647, so the antibody complex generation is unnecessary.
   2. Prepare the antibody staining solution as mentioned in Supplementary File 1.

6. RI matching

1. Immerse transparent organs in 5 mL of 50% water-diluted CUBIC-R+ solution (see Table of Materials) at RT (ON) with gentle shaking (5 x g) in a 50 mL conical tube. Keep tubes in a standing position during the entire RI matching step.
   NOTE: Recycled CUBIC R+ solutions from previous experiments could be reused (up to four times) in this step. To prepare CUBIC-R+ solution, dissolve 45% of 2,3-Dimethyl-1-phenyl-5-pyrazolone (Antipyrine), 30% of Nicotinamide or N-Methylnicotinamide, and 0.5% of N-butyldiethanolamine in double-distilled water or use the commercial CUBIC-R+ buffer (see Table of Materials).
   CAUTION: CUBIC-R+ causes skin irritation, serious eye irritation, and damage to organs. Wear protective gloves, and ensure eye and face protection. Do not breathe dust, fumes, gas, mist, or vapors. Dispose of to an approved waste disposal plant.
2. Replace 50% CUBIC-R+ with 5 mL of 100% CUBIC-R+ and incubate with gentle shaking (5 x g) at RT for 2 days. Then, transfer the tissues onto a stack of lint-free wipes and carefully turn the tissues to remove CUBIC-R+ solution from the organ surfaces for 5 min.

7. Mounting

1. After drying the tissues, transfer them to 5 mL of Mounting Solution (RI = 1.520) (see Table of Materials) in a six-well cell culture plate and incubate them (ON) at RT. Frequently turn the tissues to eliminate air bubbles, especially on the surfaces of the organs.
   NOTE: Organs can be stored for more than 6 months in Mounting Solution or CUBIC-R+.
2. Adhere the tissues to the microscope sample holder by using cyanoacrylate-based gel glue.
3. Immerse the samples in the microscope quartz cuvette filled with 120 mL of Mounting Solution and image them transversally to their longitudinal axis. Adhere the heart with the apex and the aorta horizontally aligned.
   ​NOTE: Cleared and mounted organs can be sectioned with a vibratome and imaged at high magnification by confocal microscopy. Embed the organs in 2% agarose and cut sections of 100-500 µm. Organs can also be manually sectioned with a sharp blade to produce thick-tissue sections (>1000 µm). After sectioning, place the slices into glass-bottom 35 mm Petri dishes, mount using the same mounting solution, seal with nail polish, and image with a confocal microscope.

8. Image acquisition

1. Image the mounted samples with a light-sheet microscope (see Table of Materials). Set magnification and step size between individual slices to 3 µm, and use right and left light sheet lasers with 5 µm thicknesses and 100% width. Set the exposure time constant at 50-100 ms, and adjust the laser power from 10% to 80% depending on the fluorescence signal intensity.
   1. For the co-detection of tdTomato-expressing parasites and DiR-stained dormant amastigotes (Figure 2C), use red (Ex/Em 561/620-660 nm) and infra-red (Ex/Em 785/845-55 nm) channels, respectively. For the co-detection of T cells and tdTomato-expressing parasites in CD8 reporter mouse (Figure 2D), use green (Ex/Em 488/525-50 nm) and red channel, respectively.
   2. For the coinfections assays (Figure 2E), as well as for the co-detection of parasites expressing GFP in nuclei reporter mice (Figure 2F), use the green and red channels, respectively. For the detection of tdTomato-expressing parasites, nuclear dye, and heart vasculature (Figure 3B,C), use the red, green, and far-red (Ex/Em 640/680-730 nm) channels, respectively.
   3. Detect anti-RFP antibodies with the far-red channel and anti-GFP nanobodies using the green channel (Figure 3D).
2. Convert the acquired TIFF image stacks and 3D reconstruct the organs using Imaris v9.7.2 software (see Table of Materials).

9. Surface reconstruction and quantification with Imaris software

1. Select surfaces detection algorithm tool and initiate the wizard in the image analysis software.
2. Perform an initial analysis in a 3D region of interest (randomly selected) and then apply it to the entire 3D organ reconstruction. After choosing a region of interest (ROI), select the channel to be analyzed, uncheck the smooth button, and select background subtraction.
   NOTE: Background subtraction calculates a unique local background value for every voxel and subtracts this from the original pixel value. The diameter of the largest sphere which fits into the object must be up to 200 µm for T. cruzi-infected cells, 10 µm for T cells, and 5 µm for individual parasites.
3. Use the histogram to adjust the classification and the filter drop-down list to select the measurements for classification.
   NOTE: Use the histogram to adjust the classification: ellipticity (oblate) and sphericity measurements are recommended.
4. Finalize the wizard, press statistics button, and retrieve the total number of parasite-infected cells or T cells as the number of disconnected components per time point.

Results

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 ...

Discussion

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, FLASH^34...

Materials

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