This research was supported by the Brain Research Program through the National Research Foundation (NRF) funded by the Korean Ministry of Science, ICT, and Future Planning (NRF-2015M3C7A1028790).

Animal experiments must conform to all relevant governmental and institutional regulations.
1. Preparation of Reagents
<ol>
	<li>For the hydrogel monomer solution (A4P0): Add 20 g of acrylamide to 450 mL of 0.1x phosphate-buffered saline (PBS) and adjust the volume to 500 mL with 0.1x PBS. 
	CAUTION: Acrylamide monomers are toxic. Perform all procedures in a fume hood with personal protective equipment, including a face shield, a laboratory coat, gloves, and closed-toe shoes.
	<ol>
		<li>Immediately prior to use, add 100 mg of 2,2&#39;-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride to 40 mL of hydrogel monomer solution (A4P0) in a 50 mL conical tube under a fume hood.</li>
	</ol>
	</li>
	<li>For the electrophoretic tissue clearing (ETC) buffer: Add 40 g of sodium dodecyl sulfate (SDS) and 12.37 g of boric acid to 800 mL of deionized H2O (dH2O). Adjust the pH to 8.5 with NaOH and adjust the volume to 1 L with additional dH2O. 
	CAUTION: SDS is a harmful chemical; therefore, handle it with care.</li>
	<li>For the CUBIC-mount solution: Add 250 g of sucrose, 125 g of urea, and 125 g of N,N,N&#39;,N&#39;-tetrakis(2-hydroxypropyl)ethylenediamine to 150 mL of dH2O and bring the volume to 500 mL with dH2O.</li>
</ol>
2. Sample Preparation &#38; Fixation
<ol>
	<li>Euthanasia of the mouse.
	<ol>
		<li>Prepare the alfaxalone (1 mg/kg) and xylazine (0.5 mg/kg) in the same syringe.</li>
		<li>Intraperitoneally inject the mixture of alfaxalone and xylazine and allow 3 min for the mouse to become unconscious.</li>
		<li>Wait until the anaesthetized mouse no longer responds to painful stimuli, such as a tail pinch, before proceeding. 
		CAUTION: Follow appropriate institutional guidelines for handling animals.</li>
	</ol>
	</li>
	<li>Transcardially perfuse the mouse with 100 mL of 0.9% NaCl (pH 7.4-7.5) solution containing heparin (100 U/mL) to exsanguinate, followed by 100 mL of 4% paraformaldehyde (PFA) in 1x PBS (pH 7.4)14. 
	CAUTION: PFA solution is toxic. The preparation of PFA solution and all subsequent handling must be conducted in a fume hood and with personal protective equipment.</li>
	<li>Cut off the mouse head, open up the skin, and break the skull between the eyes using small scissors.</li>
	<li>Remove pieces of the skull using small, curved forceps.</li>
	<li>Take out the brain or desired organs.</li>
	<li>Incubate the brain and organs in post-fix 4% PFA solution at 4 &#176;C overnight. 
	NOTE: For specific-region tissue clearing, dissect the specific region or trim the tissue after tissue fixation. 
	CAUTION: PFA solution is toxic. The transcardial perfusion and all subsequent mouse handling must be conducted in a fume hood and with personal protective equipment.</li>
</ol>
3. Hydrogel Monomer Infusion and Polymerization
<ol>
	<li>Incubate the fixed organs in A4P0 hydrogel monomer solution at 4 &#176;C for 12-24 h with gentle shaking.</li>
	<li>Hydrogel monomer-infused tissue polymerization.
	<ol>
		<li>Transfer the sample to a 10 mL round-bottom tube with 5 mL of hydrogel monomer solution. Wrap the top of the round-bottom tube with Parafilm.</li>
		<li>Remove the oxygen from the round-bottom tube containing the hydrogel-infused whole mouse brain sample by bubbling nitrogen through the liquid for 1 min. Quickly and tightly close the sample-containing tube.</li>
	</ol>
	</li>
	<li>Transfer the tube to a water-bath (37 &#176;C) for 2 h.</li>
	<li>Wash the polymerized sample briefly with 0.1x PBS to remove excess hydrogel. 
	CAUTION: Hydrogel monomers are toxic. To avoid skin contact with hydrogel monomer, perform all procedures in a fume hood and with personal protective equipment, including a face shield, a laboratory coat, and gloves. 
	NOTE: Polymerized tissues can be stored in 0.1x PBS containing sodium azide for more than 2 weeks at 4 &#176;C. 
	CAUTION: Sodium azide is highly and acutely toxic. Perform all procedures in a fume hood and with personal protective equipment, including a face shield, a laboratory coat, and gloves.</li>
</ol>
4. Electrophoretic Tissue Clearing (ETC)
<ol>
	<li>Transfer the polymerized sample to a tissue container and place the tissue container in the ETC chamber.</li>
	<li>Fill the ETC chamber with ETC buffer using peristatic pump.</li>
	<li>Set the ETC conditions and run the ETC. Use the following settings.
	<ol>
		<li>For ETC settings for whole organs, use the following settings: (1.5 amp), temperature (37 &#176;C), running time (6.0 h), pump speed (30 rpm).</li>
		<li>For ETC settings for 1- to 2-mm thick mouse brain slices, use the following settings: current (1.5 amp), temperature (37 &#176;C), running time (2.0 h), pump speed (30 rpm).</li>
	</ol>
	</li>
	<li>Transfer the cleared sample to a 50-mL conical tube with 45 mL of 0.1x PBS. Wash the cleared sample several times with 0.1x PBS until no bubbles are seen when the tube is briefly shaken (to confirm the complete removal of SDS).</li>
</ol>
5. Immunolabeling of ACT-processed Tissues
<ol>
	<li>For mouse whole brain: Incubate the sample in a 3-mL volume of antibody solution containing 1x PBS, 6% bovine serum albumin(BSA), 0.1% Triton X-100, and 0.01% sodium azide (antibody dilution solution) with a dilution factor of 1/500 for tyrosine hydroxylase (TH) antibody for 4 days at 37 &#176;C with mild shaking. Replace the antibody solution at the end of day 2.
	<ol>
		<li>Wash the sample in a 50-mL conical tube with 45 mL of 0.1x PBS for 3 - 5 h and change the buffer every hour.</li>
		<li>Incubate the sample in a 3-mL volume of antibody dilution solution with a dilution factor of 1/500 for donkey anti-rabbit 488 secondary antibody for 4 days at 37 &#176;C with mild shaking. Replace the diluted antibody solutions on day 2.</li>
	</ol>
	</li>
	<li>For 1- to 2-mm thick sliced brain tissue: Incubate the sample overnight or for up to 1 day in a 0.5- to 1-mL volume of antibody dilution solution with a dilution factor of 1/500 for collagen type IV antibody at 37 &#176;C with mild shaking.
	<ol>
		<li>Wash the sample in a 15-mL conical tube with a 12-mL volume of 0.1x PBS for 1-2 h. Change the buffer every hour.</li>
		<li>Incubate the sample in a 0.5- to 1-mL volume of antibody dilution solution with a dilution factor of 1/500 for Cy3-conjugated anti-rabbit secondary antibody overnight or for up to 1 day at 37 &#176;C with mild shaking.</li>
	</ol>
	</li>
	<li>Wash the sample in 0.1x PBS for 3 - 5 h; change the buffer every hour.</li>
	<li>Prior to imaging, incubate the sample in an appropriate amount of CUBIC-mount solution for 1 h at room temperature with gentle shaking. Replace with fresh CUBIC-mount solution and incubate for an additional 1 h. 
	NOTE: 76 type of antibodies worked well in mouse ACT-processed tissue, including 31 monoclonal antibodies14. Pre-labeled tissue can be combined with the immunolabeling of two more fluorochromes in ACT-processed tissue.</li>
</ol>
6. Immunolabeling of Dense Tissues (PRESTO)
<ol>
	<li>c-PRESTO 
	NOTE: c-PRESTO is suitable for small-sized samples, such as whole testis, whole kidney, or small sections of larger organs.
	<ol>
		<li>Transfer the sample into a 1.5-mL tube, add 500 &#181;L of antibody dilution solution with a dilution factor of 1/500 for collagen type IV antibody, and centrifuge the tube at 600 &#215; g for 2 h.</li>
		<li>Wash the stained sample with 0.1x PBS by centrifugation at 600 &#215; g for 30 min.</li>
		<li>Add 500 &#181;L of antibody dilution solution with a dilution factor of 1/500 for Cy3-conjugated anti-rabbit secondary antibody and centrifuge at 600 &#215; g for 2 h.</li>
		<li>Wash the stained sample with 0.1x PBS by centrifugation at 600 &#215; g for 30 min.</li>
	</ol>
	</li>
	<li>s-PRESTO 
	NOTE: s-PRESTO is suitable for larger tissues.
	<ol>
		<li>Prepare a syringe (30-mL volume) and connect it to a 3-way valve (Figure 1A). Glue the valve with a glue gun for the high-pressure conditions (Figure 1B).</li>
		<li>Transfer the sample into the 3-way valve-connected syringe in the closed-valve position.</li>
		<li>Add 5 - 7 mL of antibody dilution solution with a dilution factor of 1/500 for collagen type IV antibody. Release the antibody solution into the sample by opening the valve.</li>
		<li>Set the syringe piston to the 17-mL position to provide sufficient room for infusion/withdrawal movement. This can be done in the open-valve position. Close the 3-way valve after the syringe setting is finished.</li>
		<li>Put the syringe containing the sample on a syringe pump. Set the syringe pump conditions to an infusion/withdrawal volume of 10 mL/min and a 4-min pause time on continuous cycle mode (Figure 1C). 
		NOTE: The pump infuses until it reaches the target volume (10 mL), and then the direction of flow changes after a brief pause (4 min).</li>
		<li>Run the syringe pump for 3 - 24 h at room temperature (Figure 1F).</li>
		<li>Open the 3-way valve and replace the solution with 0.1x PBS.</li>
		<li>Wash the stained sample twice with 0.1x PBS for 1 h each time using the syringe pump.</li>
		<li>Change with antibody dilution solution containing Cy3-conjugated anti-rabbit secondary antibody (dilution factor of 1/500) and run the syringe pump for 3 - 24 h.</li>
		<li>Open the 3-way valve and replace the solution with 0.1x PBS.</li>
	</ol>
	</li>
	<li>Prior to imaging, incubate the stained sample in an appropriate amount of CUBIC-mount solution for 1 h at room temperature with gentle shaking. Replace with fresh CUBIC-mount solution and incubate for an additional 1 h.</li>
</ol>
7. Imaging
<ol>
	<li>Place the labeled tissue in a confocal dish and add CUBIC-mount solution until the tissue is covered. Place a coverslip on the tissue. Use a confocal microscope to image the tissue14 under the 10X objective.</li>
</ol>

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T., Fujimoto, S., Imai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeDB:+a+simple+and+morphology-preserving+optical+clearing+agent+for+neuronal+circuit+reconstruction.">SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.</a> Nat Neurosci. 16, 1154-1161 (2013).</li><li>Ert&amp;#252;rk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO.">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nat Protoc. 7, 1983-1995 (2012).</li><li>Hama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nat Neurosci. 14, 1481-1488 (2011).</li><li>Renier, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iDISCO:+a+simple,+rapid+method+to+immunolabel+large+tissue+samples+for+volume+imaging.">iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.</a> Cell. 159, 896-910 (2014).</li><li>Susaki, E. 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A., Schultz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+clearing+for+optical+anatomy.">Tissue clearing for optical anatomy.</a> Angew Chem Int Edit. 52, 10949-10951 (2013).</li><li>Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues.">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nat Protoc. 9, 1682-1697 (2014).</li><li>Lee, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACT-PRESTO:+Rapid+and+consistent+tissue+clearing+and+labeling+method+for+3-dimensional+(3D)+imaging.">ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging.</a> Sci Rep. 6, 18631 (2016).</li><li>Murray, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+Proteomic+Imaging+for+High-Dimensional+Profiling+of+Intact+Systems.">Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems.</a> Cell. 163, 1500-1514 (2015).</li></ol>

The authors declare that they have no competing financial interests.

Poor fixation or hydrogel immersion into tissues can cause the loss of proteins and the distortion of tissue during the tissue clearing process. The whole adult mouse brain should be incubated in 4% paraformaldehyde overnight, followed by immersion in a minimum volume of 20 mL of hydrogel monomer solution for 12 - 18 h with gentle shaking. For large tissues, including human tissue such as brain slices and spinal cord, extended soaking time in the hydrogel monomer solution is required. The ACT protocol is easily applicable to the clearing of fixed tissues because the tissue fixation and polymer infusion steps are separated. After tissue clearing, human tissues were well-stained with several antibodies. Note that the ACT clearing technique is not compatible with alcohol fixatives, such as ethanol and methanol.
The tissue-hydrogel polymerization step is also critical to produce good-quality tissue following the clearing process. Because oxygen inhibits the polymerization of acrylamide, it should be removed by degassing the tissue-containing solution under a nitrogen gas infusion system. Alternatively, de-oxygenation can be executed using a vacuum chamber with a heat block for 23 h.
The clearing rate is dependent upon numerous factors, including the organ size, the contents of lipids or ECM fibrous proteins, the condition of fixation, etc. Most adult mouse tissues can be cleared by ETC overnight. However, there is a risk of unnecessary over-clearing and tissue swelling; thus, the differences in clearing time are of important consideration. Tissue-clearing conditions should be empirically determined. Importantly, the contents of the ECM may affect the appearance of the cleared tissue. The color of the tissues remains white or opaque, even after the ACT in ETC buffer, because ACT does not clear dense protein fibers. However, when these organs were immersed in RI matching solution, they became transparent.
The rate of tissue swelling appears to be dependent upon the ECM contents of the tissues, and soft organs, such as the brain, exhibit a greater swelling ratio than dense organs. Because increased tissue swelling will help the tissue-clearing procedure, ACT routinely utilizes distilled water-based buffer in most cases. In some cases, when tissue swelling or transient deformity is not desirable, such as in embryos, buffer containing 0.1x PBS can be applied for high-resolution imaging. It should be also noted that higher salt concentrations in the buffer could cause the shrinkage of tissues.
The ACT method can clarify whole organs and even the whole body of a mouse14. However, deep-tissue imaging of transparent tissue requires special microscopes and objectives. Thus, brain tissue 1- to 2-mm thick is most effective for imaging with a conventional confocal microscope. In our hands, removal of the labeled antibodies from ACT-processed tissues is antibody-dependent, and rounds of different antibody labeling are not recommended. Several antibodies, such as TH and GFAP, worked especially well for whole-brain immunolabeling14. On the other hand, some antibodies, such as Tuj1 or Map2, often labeled only the surface of the tissues. This might be improved by other methods, such as SWITCH15. Another potential limitation is that it might be difficult to preserve fine protein structures in ACT-processed tissue because of the tissue swelling and shrinkage during the tissue-clearing step.
The PRESTO technique is applicable to a wide variety of tissue-labeling techniques. For example, in tissue-transparent methods using pre-labeled tissue, such as SeeDB4 and iDISCO7, immunolabeling of dense tissue requires incubation periods longer than several days to weeks. However, PRESTO can shorten the incubation time to several hours, enabling the completion of the entire process within a day with 1-mm thick dense tissue. PRESTO can enhance the efficacy of deep-labeling thick, dense tissues, or even un-cleared thick tissues. Because PRESTO uses a table-top centrifuge or syringe pump and does not require any special equipment, it is relatively easy to implement this technique with routine lab procedures.

One of the challenges in neuroscience is the visualization of neuronal circuit wiring and individual cells within intact brain tissue. Until recently, demonstrating the connections between neurons in this way required 1) serial sectioning of tissues; 2) molecular labeling of specific targets, such as axons or proteins; and 3) visualization by 3D reconstruction of the whole brain via computational registering or alignment of 2D serial images1. These steps are laborious, require a great deal of time, and are liable to lose information during sectioning and labeling, making neuronal network mapping exceedingly difficult. However, many methods that allow for the visualization of intact tissue without sectioning have been developed. Biological tissues can be made optically transparent by tissue-clearing techniques2-10. One of the major methods is to reduce refractive index differences between the intact tissue and the immersion solution in order to reduce light scattering in the intact tissue, thus rendering the tissue transparent and enabling the observation of deep structures. Some types of immersion solutions have hydrophobic properties, which result in fast fluorescent quenching during the dehydration procedure. Therefore, those methods are not compatible with fluorescent imaging over a long period of time11,12. Instead of hydrophobic reagents, other methods use hydrophilic reagents for tissue clearing, such as SeeDB4 and Scale6, which maintain the structural information and fluorescence of the biological tissue4,6,8. However, macromolecules, including antibodies, cannot reach the core of intact tissue by diffusion alone. Therefore, the pre-labeling of target molecules in deep areas of densely-packed tissues is technically challenging.
In a recently-developed tissue-clearing method, CLARITY3, a hydrogel-embedded biological tissue is formed with acrylamide, and lipids are removed by electrophoresis under sodium dodecyl sulfate (SDS)-containing solution. The sample is then immersed in a solution with a matching reflective index to reduce light scattering3. The lipid removal system was later modified in advanced CLARITY13 and PACT (passive clarity technique)10. After polymerization, acrylamide chains crosslink with proteins, forming hydrogel-tissue. Lipid components cannot crosslink with acrylamide; thus, lipids can be eliminated by electrophoresis under the SDS-containing buffer. Through the active elimination of lipids, brain tissue markedly increases in transparency9. However, these methods do not address the impossibility of deep-labeling by free diffusion. To overcome this limitation, techniques for the active transport of reagents into the deepest parts of thick tissues are required.
Although CLARITY allows tissue clearing and deep-tissue visualization, it is not an easy or fast procedure. It can take several weeks to clear a whole mouse brain7,14. Rapid clearing of tissues is essential for the application of such methods to basic or clinical laboratory settings for life science research or volume diagnosis. The current protocol provides a simplified process for biological tissue clearance and subsequent protein detection by pressure-assisted delivery immuno-staining. It is suitable for high-resolution volume imaging of large filed and 3D data processing systems through combination with imaging methods.

<table><tbody><tr><td>4% Paraformaldehyde</td><td>Lugen SCI&amp;nbsp;</td><td>LGB-1175-4B</td><td>sample fixation&amp;nbsp;</td></tr><tr><td>Acrylamide</td><td>Affymetrix</td><td>75820</td><td>Hydrogel monomer solution&amp;nbsp;</td></tr><tr><td>2,2&amp;rsquo;-Azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride</td><td>Wako Pure Chemical Industries&amp;nbsp;&amp;nbsp;</td><td>VA-044</td><td>Hydrogel monomer solution&amp;nbsp;</td></tr><tr><td>Boric acid</td><td>Affymetrix</td><td>76324</td><td>ETC buffer</td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Affymetrix</td><td>18220</td><td>ETC buffer</td></tr><tr><td>Sodium hydroxide pellets</td><td>Junsei chemical</td><td>1310-73-2</td><td>ETC buffer</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Santa Cruz Biotechnology</td><td>sc-2323A</td><td>Immunolabeling</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Immunolabeling</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S2002</td><td>Immunolabeling</td></tr><tr><td>Tyrosine hydroxylase (TH)</td><td>Millipore</td><td>AB152</td><td>Antibody/immunolabeling</td></tr><tr><td>Alexa Fluor Cy3 Donkey anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>711-165-152</td><td>Secondary antibody/ immunolabeling</td></tr><tr><td>Alexa Fluor 488 Donkey anti-Rabbit IgG (H+L)</td><td>Life Technologies - Molecular Probes</td><td>A21206</td><td>Secondary antibody/ immunolabeling</td></tr><tr><td>Confocal dish</td><td>SPL lifesciences</td><td>101350</td><td>Imaging</td></tr><tr><td>Confocal microscope</td><td>Leica</td><td>SP8</td><td>Imaging</td></tr><tr><td>Sucrose</td><td>Junsei chemical</td><td>31365-0301</td><td>CUBIC-mount solution&amp;nbsp;</td></tr><tr><td>Urea</td><td>Affymetrix</td><td>23036</td><td>CUBIC-mount solution&amp;nbsp;</td></tr><tr><td>&lt;em&gt;N,N,N&amp;rsquo;,N&amp;rsquo;&lt;/em&gt;-tetrakis(2-hydroxypropyl)ethylenediamine</td><td>Sigma-Aldrich</td><td>122262</td><td>CUBIC-mount solution&amp;nbsp;</td></tr><tr><td>ECT chamber</td><td>Logos Biosystems, Inc.</td><td>C10101</td><td>ETC system</td></tr><tr><td>ECT chamber controller</td><td>Logos Biosystems, Inc.</td><td>C10201</td><td>ETC system</td></tr><tr><td>Temperature probe</td><td>Logos Biosystems, Inc.</td><td>C12101</td><td>ETC system</td></tr><tr><td>Peristatic pump</td><td>Baoding longer precision pump Co., Ltd</td><td>YZ1515X</td><td>ETC system</td></tr><tr><td>Buffer reservoir</td><td>Logos Biosystems, Inc.</td><td>C10401</td><td>ETC system</td></tr><tr><td>Tissue container&amp;nbsp;</td><td>Logos Biosystems, Inc.</td><td>C12001</td><td>ETC system</td></tr><tr><td>Container holder for 1 tissue container</td><td>Logos Biosystems, Inc.</td><td>C12002</td><td>ETC system</td></tr><tr><td>Mouse brain slice holder</td><td>Logos Biosystems, Inc.</td><td>C12004</td><td>ETC system</td></tr><tr><td>Whole rat brain holder</td><td>Logos Biosystems, Inc.</td><td>C12007</td><td>ETC system</td></tr><tr><td>Peristaltic pump tubing</td><td>Logos Biosystems, Inc.</td><td>C12104</td><td>ETC system</td></tr><tr><td>Tabletop-centrifuge</td><td>Hanil science industrial Co., Ltd</td><td>MICRO 12</td><td>c-PRESTO</td></tr><tr><td>Syringe pump</td><td>Baoding longer precision pump Co., Ltd</td><td>LSP02-1B</td><td>s-PRESTO</td></tr><tr><td>Syringe (30 mL)</td><td>Korea vaccine Co., Ltd.</td><td>KV-S30</td><td>s-PRESTO</td></tr><tr><td>3-way stopcock</td><td>Hyupsung medical Co.,Ltd.</td><td>HS-T-01N</td><td>s-PRESTO</td></tr></tbody></table>

act-presto: biological tissue clearing and immunolabeling methods for volume imaging

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in biology. Transitional methods require a substantial amount of time and effort to obtain a 3D image and including sectioning the intact tissue, immunolabeling, and imaging serially-sectioned tissue, which produces a loss of information at each step of the process. In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization has been restricted to the labeling of proteins. However, currently available protocols for organ clearing require a considerably long process time, making it difficult to implement tissue clearing techniques in the lab. We recently established a rapid and highly-reproducible protocol termed ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs), which allows for tissue clearance within several hours. Moreover, ACT-PRESTO enables rapid immunolabeling with conventional methods and accelerates antibody penetration into the deep layer of densely-formed, thick specimens by applying pressure or convection flow. We describe how to prepare tissues, how to clear by lipid removal using electrophoresis, and how to immuno-stain by a pressure-assisted delivery. The rapidity and consistency of the protocol will expedite the performance of 3D histological research and volume-based diagnoses.

ACT-tissue Clearing
One important factor affecting tissue clearing is tissue fixation. PFA fixation and acrylamide infusion are separate steps in this protocol. After the tissue-hydrogel polymerization step, the free A4P0 solution is not polymerized, although tissue-infused hydrogels are cross-linked with endogenous macromolecules. Therefore, no gel should form outside of the tissues (Figure 2A). The ACT procedure results in less cross-linking between proteins and acrylamide compared to the CLARITY protocol. Accordingly, the tissue-hydrogel sample is more porous. This feature enables the fast extraction of lipids and the diffusion of macromolecules. Sections of mouse brain 1- to 2-mm thick were sufficiently cleared within 1 - 2 h (Figure 2B), and 5 - 6 h of ETC was sufficient for clearance of whole mouse brains (Figure 2C). Hydrogel-mediated tissue clearing induced the expansion of the tissue after the ETC step, but the tissue returned to its original size in reflective index-matched solution. The tissue-hydrogel sample formed in the ACT procedure is highly porous, and thus, small compounds and macromolecules could penetrate efficiently and diffuse easily (Figure 3). After a 2-h incubation of 1-mm brain slices with tyrosine hydroxylase (TH) and collagen type IV antibody, a subsequent 2-h incubation with a secondary antibody was sufficient to label dopaminergic neurons at a 500-&#181;m depth (Figure 3).
PRESTO-tissue Immunolabeling
Dense organs generally have high concentrations of ECM fibers, which affects the penetration of antibodies into the tissues. Thus, antibodies penetrated to a depth of only 20-30 &#181;m in dense tissues, such as kidney tissue, after 12 h of incubation. To overcome this limitation, we designed active macromolecule penetration methods that enable the delivery of reagents into deep tissues, namely PRESTO (pressure related efficient and stable transfer of macromolecules into the organs) (Figure 4). Compared to the static incubation of ACT-processed kidney tissue with antibody solutions, the application of centrifugal forces (600 x g) with a table-top centrifuge (centrifugal PRESTO, c-PRESTO) greatly improved antibody delivery into deep tissue (Figure 5). When we applied the c-PRESTO procedure to dense tissues, 3 h of incubation was sufficient to label 250- to 300-&#181;m deep structures. To improve the tissue distortion without significant tissue damage, we also designed a machine that provides convection flow with a syringe pump (syringe PRESTO, s-PRESTO). This process similarly improved antibody penetration compared to passive diffusion (Figure 5).
<img alt="Figure 1" src="/files/ftp_upload/54904/54904fig1.jpg" /> 
Figure 1. Preparation of the s-PRESTO apparatus. A. A syringe (30 mL) and three-way stopcock. B. The three-way stopcock connected and glued to the syringe. C. The syringe containing the sample and antibody solution, and the syringe placed on the syringe pump. D. The syringe pump and working condition setup. E. The pump infuses a solution containing the labeling reagents into the specimen. When the designated volume is reached, the pumping direction changes and withdraws the solution after a designated period of time. <a href="http://cloudflare.jove.com/files/ftp_upload/54904/54904fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54904/54904fig2.jpg" /> 
Figure 2. ACT tissue clearing of mouse brain. A. After polymerization, free A4P0 solutions are not polymerized; tissue-infused hydrogels are gelated by cross-linking with endogenous macromolecules. B. 1- to 2-mm thick brain slices were cleared for 1 - 2 h, and the extent of expansion and size recovery in reflective index (RI)-matching solution were recorded. C. The thickness of the whole mouse brain is about 0.8 cm, and 5 - 6 h of ETC was sufficient to completely clear the brain. After 3 h of ETC, there was a significant amount of non-cleared tissue. By 6 h of ETC, however, clearing of the entire brain had occurred. The color of tissues after ACT in the ETC buffer is white or opaque. By RI adjustment, tissues become transparent. <a href="http://cloudflare.jove.com/files/ftp_upload/54904/54904fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54904/54904fig3.jpg" /> 
Figure 3. Immunolabeling with ACT-cleared mouse brain tissue. Images of ACT-processed 1-mm mouse brain slices showing mouse brain cortex stained with an antibody to collagen type IV and part of the midbrain stained with an antibody to tyrosine hydroxylase (TH). Scale bar, 100 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54904/54904fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54904/54904fig4.jpg" /> 
Figure 4. PRESTO immunolabeling methods. Antibodies cannot penetrate into dense tissues by diffusion. PRESTO immunolabeling methods were designed to actively infuse macromolecules and to deliver reagents into dense tissues. The application of centrifugal forces using a standard table-top centrifuge (centrifugal PRESTO, c-PRESTO) markedly facilitated the delivery of antibodies. Appling convection flow with a syringe pump (syringe PRESTO, s-PRESTO) improved antibody penetration. <a href="http://cloudflare.jove.com/files/ftp_upload/54904/54904fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54904/54904fig5.jpg" /> 
Figure 5. PRESTO immunolabeling of ACT-cleared dense tissue. For c-PRESTO, tissues were centrifuged at 600 x g for 3 h using a standard table-top centrifuge in order to accelerate the penetration of primary and secondary antibodies. For s-PRESTO, a syringe pump was used to deliver the antibodies. Mouse organs, such as the kidneys and liver, were labeled with collagen type IV. Compared to conventional methods, 3 h of c- or s-PRESTO markedly enhances the labeling depth. In the kidney and liver, the depth of the Z-axis reaches 300 - 350 &#181;m or deeper in PRESTO samples (right), while control samples are labeled at a depth of only 40 - 50 &#181;m (left). A 3D reconstructed image was obtained with a confocal microscope. Scale bar, 100 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54904/54904fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging at JoVE.com

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs) enables rapid, efficient, but inexpensive tissue clearing and rapid antibody penetration into cleared tissues by passive diffusion or pressure-assisted delivery. Using this method, a wide range of tissues can be cleared, immunolabeled by multiple antibodies, and volume-imaged.

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in ...

act-presto-biological-tissue-clearing-immunolabeling-methods-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.0K Views.  Korea University College of Medicine. ACT-PRESTO (active clarity technique–pressure related efficient and stable transfer of macromolecules into organs) enables rapid, efficient, but inexpensive tissue clearing and rapid antibody penetration into cleared tissues by passive diffusion or pressure-assisted delivery. Using this method, a wide range of tissues can be cleared, immunolabeled by multiple antibodies, and volume-imaged.

Video: ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Developmental Biology

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.

To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen.

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact ...

Neuroscience

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

Medicine

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 &#181;m (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY&#160;can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei.
Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases.
We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of ...

High-Resolution 3D Reconstruction of Human Brain with SHORT and LSFM

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with light-sheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi-labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high-throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.

Author Spotlight: Advancing 3D Cytoarchitecture Analysis - Rapid Volumetric Reconstruction of the Human Brain 

The present protocol provides a step-by-step procedure for rapid and simultaneous optical clearing, muti-round labeling, and 3D volumetric reconstruction of tens of postmortem human brain sections by combining the (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT tissue transformation technique with light-sheet fluorescence microscopy imaging in a routinely high-throughput protocol.

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its ...

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 &#181;m to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffin-embedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 &#181;m depth) by confocal microscopy.

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 &#181;m to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and ...