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>

<ol>
	<li>Micheva, K. D., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array+tomography:+a+new+tool+for+imaging+the+molecular+architecture+and+ultrastructure+of+neural+circuits.">Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits.</a> Neuron. 55, 25-36 (2007).</li><li>Dodt, H. -. U., 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=Ultramicroscopy:+three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain.">Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.</a> Nat Methods. 4, 331-336 (2007).</li><li>Chung, K., 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=Structural+and+molecular+interrogation+of+intact+biological+systems.">Structural and molecular interrogation of intact biological systems.</a> NATURE. 497, 332-337 (2013).</li><li>Ke, M. -. 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. 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=Whole-brain+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis.">Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.</a> Cell. 157, 726-739 (2014).</li><li>Tainaka, K., 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=Whole-body+imaging+with+single-cell+resolution+by+tissue+decolorization.">Whole-body imaging with single-cell resolution by tissue decolorization.</a> Cell. 159, 911-924 (2014).</li><li>Yang, B., 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=Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158, 945-958 (2014).</li><li>Kim, S. -. 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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 border="0" cellpadding="0" cellspacing="0" width="813">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">4% Paraformaldehyde</td>
			<td style="width:175px;">Lugen SCI&#160;</td>
			<td style="width:201px;">LGB-1175-4B</td>
			<td style="width:191px;">sample fixation&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Acrylamide</td>
			<td style="width:175px;">Affymetrix</td>
			<td style="width:201px;">75820</td>
			<td style="width:191px;">Hydrogel monomer solution&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">2,2&#8217;-Azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride</td>
			<td style="width:175px;">Wako Pure Chemical Industries&#160;&#160;</td>
			<td style="width:201px;">VA-044</td>
			<td style="width:191px;">Hydrogel monomer solution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Boric acid</td>
			<td style="width:175px;">Affymetrix</td>
			<td style="width:201px;">76324</td>
			<td>ETC buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td style="width:175px;">Affymetrix</td>
			<td style="width:201px;">18220</td>
			<td>ETC buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Sodium hydroxide pellets</td>
			<td style="width:175px;">Junsei chemical</td>
			<td style="width:201px;">1310-73-2</td>
			<td>ETC buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:175px;">Santa Cruz Biotechnology</td>
			<td style="width:201px;">sc-2323A</td>
			<td>Immunolabeling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Triton X-100</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:201px;">T8787</td>
			<td>Immunolabeling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Sodium azide</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:201px;">S2002</td>
			<td>Immunolabeling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Collagen type &#8547;</td>
			<td style="width:175px;">Abcam</td>
			<td style="width:201px;">AB6586</td>
			<td>Antibody/immunolabeling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tyrosine hydroxylase (TH)</td>
			<td style="width:175px;">Millipore</td>
			<td style="width:201px;">AB152</td>
			<td>Antibody/immunolabeling</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Alexa Fluor Cy3 Donkey anti-Rabbit IgG (H+L)</td>
			<td style="width:175px;">Jackson ImmunoResearch</td>
			<td style="width:201px;">711-165-152</td>
			<td style="width:191px;">Secondary antibody/ immunolabeling</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Alexa Fluor 488 Donkey anti-Rabbit IgG (H+L)</td>
			<td style="width:175px;">Life Technologies - Molecular Probes</td>
			<td>A21206</td>
			<td style="width:191px;">Secondary antibody/ immunolabeling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Confocal dish</td>
			<td style="width:175px;">SPL lifesciences</td>
			<td style="width:201px;">101350</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Confocal microscope</td>
			<td style="width:175px;">Leica</td>
			<td style="width:201px;">SP8</td>
			<td>Imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Sucrose</td>
			<td style="width:175px;">Junsei chemical</td>
			<td style="width:201px;">31365-0301</td>
			<td>CUBIC-mount solution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Urea</td>
			<td style="width:175px;">Affymetrix</td>
			<td style="width:201px;">23036</td>
			<td>CUBIC-mount solution&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">N,N,N&#8217;,N&#8217;-tetrakis(2-hydroxypropyl)ethylenediamine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:201px;">122262</td>
			<td>CUBIC-mount solution&#160;</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:247px;">ECT chamber</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C10101</td>
			<td>ETC system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">ECT chamber controller</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C10201</td>
			<td>ETC system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Temperature probe</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C12101</td>
			<td>ETC system</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Peristatic pump</td>
			<td style="width:175px;">Baoding longer precision pump Co., Ltd</td>
			<td style="width:201px;">YZ1515X</td>
			<td>ETC system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Buffer reservoir</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C10401</td>
			<td>ETC system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Tissue container&#160;</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C12001</td>
			<td>ETC system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Container holder for 1 tissue container</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C12002</td>
			<td>ETC system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Mouse brain slice holder</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C12004</td>
			<td>ETC system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Whole rat brain holder</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C12007</td>
			<td>ETC system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Peristaltic pump tubing</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C12104</td>
			<td>ETC system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Tabletop-centrifuge</td>
			<td style="width:175px;">Hanil science industrial Co., Ltd</td>
			<td style="width:201px;">MICRO 12</td>
			<td>c-PRESTO</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Syringe pump</td>
			<td style="width:175px;">Baoding longer precision pump Co., Ltd</td>
			<td style="width:201px;">LSP02-1B</td>
			<td>s-PRESTO</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Syringe (20 ml)</td>
			<td style="width:175px;">Korea vaccine Co., Ltd.</td>
			<td style="width:201px;">KV-S20</td>
			<td>s-PRESTO</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">3-way stopcock</td>
			<td style="width:175px;">Hyupsung medical Co.,Ltd.</td>
			<td style="width:201px;">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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

Methods for the Study of the Zebrafish Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle. 
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (&gt;3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Volume Segmentation and Analysis of Biological Materials Using SuRVoS (Super-region Volume Segmentation) Workbench

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of interest. When considering the analysis of complex biological systems, the segmentation of three-dimensional image data is a time consuming and labor intensive step. With the increased availability of many imaging modalities and with automated data collection schemes, this poses an increased challenge for the modern experimental biologist to move from data to knowledge. This publication describes the use of SuRVoS Workbench, a program designed to address these issues by providing methods to semi-automatically segment complex biological volumetric data. Three datasets of differing magnification and imaging modalities are presented here, each highlighting different strategies of segmenting with SuRVoS. Phase contrast X-ray tomography (microCT) of the fruiting body of a plant is used to demonstrate segmentation using model training, cryo electron tomography (cryoET) of human platelets is used to demonstrate segmentation using super- and megavoxels, and cryo soft X-ray tomography (cryoSXT) of a mammalian cell line is used to demonstrate the label splitting tools. Strategies and parameters for each datatype are also presented. By blending a selection of semi-automatic processes into a single interactive tool, SuRVoS provides several benefits. Overall time to segment volumetric data is reduced by a factor of five when compared to manual segmentation, a mainstay in many image processing fields. This is a significant savings when full manual segmentation can take weeks of effort. Additionally, subjectivity is addressed through the use of computationally identified boundaries, and splitting complex collections of objects by their calculated properties rather than on a case-by-case basis.

Volume Segmentation and Analysis of Biological Materials Using SuRVoS Super-region Volume Segmentation Workbench

Segmentation of three-dimensional data from many imaging techniques is a major bottleneck in analysis of complex biological systems. Here, we describe the use of SuRVoS Workbench to semi-automatically segment volumetric data at various length-scales using example datasets from cryo-electron tomography, cryo soft X-ray tomography, and phase contrast X-ray tomography techniques.

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic phenomena, such as cytokinesis, mitotic spindle formation and assembly of the nucleus. Building upon early methods that imaged fixed extracts sampled at sparse time points, recent approaches image live extracts using time-lapse microscopy, revealing more dynamical features with enhanced temporal resolution. These methods usually require sophisticated surface treatments of the imaging vessel. Here we introduce an alternative method for live imaging of egg extracts that require no chemical surface treatment. It is simple to implement and utilizes mass-produced laboratory consumables for imaging. We describe a system that can be used for both wide-field and confocal microscopy. It is designed for imaging extracts in a 2-dimensional (2D) field, but can be easily extended to imaging in 3D. It is well-suited for studying spatial pattern formation within the cytoplasm. With representative data, we demonstrate the typical dynamic organization of microtubules, nuclei and mitochondria in interphase extracts prepared using this method. These image data can provide quantitative information on cytoplasmic dynamics and spatial organization.

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

We describe a method for the preparation and live imaging of undiluted cytoplasmic extracts from Xenopus laevis eggs.