Name: Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue (Video) | JoVE Journal
Uploaded: 2018-05-08T14:00:00.000+00:00
Duration: 6 min 14 s
Description: Discover psPACT and mPACT methods for rapid optical clearing of whole rodent CNS tissue without electrophoretic damage. These techniques enable efficient microscopic visualization of vasculature for neuroscience research.

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5 minutes

This work was supported by the Brain Korea 21 PLUS Project for Medical Science, Yonsei University. In addition, this work was supported by a grant from the National Research Foundation of Korea (NRF-2017R1D1A1B03030315).

All procedures have been approved by the appropriate research ethics committee at Yonsei University College of Medicine. All experimental animals are sacrificed in accordance with the guidelines of the laboratory animal care committee at Yonsei University College of Medicine.
1. Preparation of Reagents
Caution: Paraformaldehyde (PFA), acrylamide and sodium dodecyl sulfate (SDS) are toxic irritants and thus should be handled in a fume hood with appropriate personal protective equipment (PPE; lab coat, gloves, protective eyewear).
<ol>
	<li>4% acrylamide (A.A) solution (A4P0): Add 20 mL of 40% acrylamide solution to 180 mL of 0.1 M phosphate-buffered saline (PBS).</li>
	<li>0.25% VA-044 solution: Add 0.5 g of 2,2&#180;-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044) powder to 200 mL of 0.1 M phosphate-buffered saline (PBS). While the VA-044 powder can be stored at room temperature, it must be stored at 4 &#176;C upon solubilization in PBS. For best results, prepare only the amount of solution needed for the experiment.</li>
	<li>PACT cocktail solution: Add 10 g of acrylamide to 225 mL of 4% paraformaldehyde (PFA) solution and adjust the volume to 200 mL with 4% PFA. Prior to use, add 100 mg of VA-044 powder to 40 mL of mixture in a 50 mL conical tube.</li>
	<li>PACT clearing solution buffer: Add 40 g of sodium dodecyl sulfate (SDS) to 350 mL of 0.1 M PBS and adjust the volume to 500 mL with 0.1 M PBS. For mPACT clearing solution, add 0.5% &#945;-thioglycerol to the PACT clearing solution buffer.</li>
	<li>The mice used in this study were 2-week-old BALB male mice; the rats used in this study were 2 week old male SD-rats.</li>
</ol>
2. Anesthesia and Perfusion Surgery
Caution: PFA and acrylamide are toxic irritants and thus should be handled in a fume hood with appropriate PPE.
<ol>
	<li>Anesthetize the animal in a pathogen-free room with 30 mg/kg of zoletil using a 1 mL syringe with a 26-gauge needle. Monitor the animal for 5 -10 min.</li>
	<li>Once the animal has reached a surgical plane of anesthesia, use the toe-pinch response method to determine the depth of anesthesia. Confirm unresponsiveness. 
	NOTE: The following steps involving mouse and rat surgery follow a similar protocol used in a previous study11.</li>
	<li>Make a 5-6 cm incision beneath the rib cage, through the integument and abdominal wall.</li>
	<li>Use curved, blunt scissors to make an incision in the diaphragm, palpating the thoracic area to locate the site of incision beforehand.</li>
	<li>Extend the incision across the entire length of the ribcage to expose the pleural cavity.</li>
	<li>Carefully displace the lungs. Make a cut through the ribcage up to the collarbone. Using iris scissors, make a small incision to the posterior end of the left ventricle. Insert an 18-gauge blunt- or olive-tipped perfusion needle into the ascending aorta, passing through the cut ventricle.</li>
	<li>Secure the needle and prevent leakage by clamping the heart with a hemostat. Alternatively, use a modified hemostat to clamp the aorta around the needle tip.</li>
	<li>Make a large incision in the right atrium, taking care to ensure minimal damage to the descending aorta. The rat is now ready for perfusion.</li>
</ol>
3. Whole Perfusion and Dissection of Rat
Caution: PFA and acrylamide are toxic irritants and thus should be handled in a fume hood with appropriate PPE.
NOTE: The following whole perfusion steps are a similar to a protocol used in a previous study by Woo et al. (2016)10,11.
<ol>
	<li>Attach the right atrium of heart to an 18-gauge needle, taking care to not introduce any air bubbles.</li>
	<li>Using a 50 mL syringe, quickly and evenly pump 50 mL of cold 0.1 M PBS solution containing heparin (10 units/mL).</li>
	<li>Connect the 18-gauge needle to the tube of the peristaltic pump.</li>
	<li>Wash with 200 mL of cold 0.1 M PBS solution containing heparin (10 units/mL) at a circulation velocity of 10 mL/min.</li>
	<li>Fix with 250 mL of cold 4% PFA solution at a circulation velocity of 10 mL/min. The rat should be stiff at this stage.</li>
	<li>Collect and store the remaining PFA solution for disposal.</li>
	<li>For PACT, perfuse tissues with a chilled PACT cocktail solution of 4% PFA, 4% acrylamide, and 0.25% VA-044 powder with an 18-gauge needle, and then remove the head and spine. Expose the skull by making a midline incision from the neck to the nose. This additional perfusion step is unnecessary for the psPACT and mPACT methods; after fixation, immediately isolate the head and spine and expose the skull.</li>
	<li>Expose the base of the skull by removing any residual neck and spine muscle.</li>
	<li>Use rongeurs and scissors to peel away the skull. Remove the brain and spinal cord.</li>
	<li>Store the tissue in 4% PFA at 4 &#176;C; tissues can be stored for up to 1 week.</li>
</ol>
4. Hydrogel Monomer Infusion and Polymerization of the Rat and Mouse CNS
Caution: Acrylamide, SDS, &#945;-thioglycerol and PFA are irritants and thus should be handled in a fume hood and with appropriate PEE.
<ol>
	<li>PACT (Passive Clearing Technique)
	<ol>
		<li>Isolate and culture the whole CNS (brain and spinal cord) from fixed mice and rats to a 50 mL tube containing a chilled PACT cocktail solution of 4% PFA, 4% acrylamide, and 0.25% VA-044 powder and store at 4 &#176;C for 24 h. Ensure that the tissue is completely immersed in the solution.</li>
		<li>Embed the sample in nitrogen gas for 10 min using a tissue gel hybridization system connected to a nitrogen tank: Set the system to 37 &#176;C. Transfer tissues to a 50 mL tube containing fresh cold (4 &#176;C) PACT cocktail solution and connect with tube cap, then turn on the vacuum.</li>
		<li>To polymerize the hydrogel, place the tube containing the sample in a shaking incubator (150 rpm, 37 &#176;C) for 3 h, or until polymerization is complete.</li>
		<li>Using blotting paper (see Table of Materials), remove the remaining polymerized hydrogel surrounding the tissues.</li>
		<li>Transfer the tissue to a 50 mL tube containing clearing solution (8% SDS in 0.1 M PBS, pH 8.0).</li>
		<li>Place the sample in a shaking incubator set to 37 &#176;C and 150 rpm until the tissue has been cleared. On average, it takes about 20 days for the mouse CNS to achieve full clarity.</li>
	</ol>
	</li>
	<li>psPACT (process separate Passive Clearing Technique)
	<ol>
		<li>Isolate the whole CNS (brain and spinal cord) with bone cutter and scissors from PFA-fixed mice and rats on a clean bench.</li>
		<li>Transfer tissues to a 50 mL tube containing 4% PFA, and store at 4 &#176;C for 24 h. Ensure that the tissue is completely immersed in the fixative.</li>
		<li>Wash the fixed tissue for 1 h in 0.1 M PBS, and then transfer to A4P0 solution (4% acrylamide in 0.1 M PBS). Store at 37 &#176;C for 24 h.</li>
		<li>Wash the tissue for 5 min in 0.1 M PBS.</li>
		<li>Immerse the tissue in 0.25% VA-044 in 0.1 M PBS. Store at 37 &#176;C for 6-24 h, and then transfer to fresh 0.25% VA-044/PBS solution.</li>
		<li>Embed the sample in nitrogen gas for 10 min using a tissue gel hybridization system (see Table of Materials) connected to a nitrogen tank: Transfer tissues to a 50 mL tube containing fresh cold (4 &#176;C) 0.25% VA-044/PBS solution, and connect with tube cap. Turn on the vacuum.</li>
		<li>Transfer the tissue to clearing solution (8% SDS in 0.1 M PBS, pH 8.0).</li>
		<li>Incubate the sample in a shaking incubator set to 37 &#176;C and 150 rpm until the tissue has been cleared. On average, it takes approximately 17 days for mouse CNS to achieve full clarity.</li>
	</ol>
	</li>
	<li>mPACT (modified Passive Clearing Technique)
	<ol>
		<li>Isolate the whole CNS (brain and spinal cord) with bone cutter and scissors from PFA-fixed mice and rats on a clean bench.</li>
		<li>Follow steps 4.2.2 - 4.2.8 of the psPACT protocol.</li>
		<li>Transfer the tissue to clearing solution. Note that unlike the PACT and psPACT protocols, mPACT requires a clearing solution consisting of 0.5% &#945;-thioglycerol in addition to 8% SDS in 0.1 M PBS, pH 8.0. &#945;-thioglycerol is an un-browning agent that helps clear tissue more rapidly and effectively. Ensure that the tissue is fully immersed in the solution.</li>
		<li>Place the sample in a shaking incubator set to 37 &#176;C and 150 rpm until the tissue has been cleared. On average, it takes approximately 2 weeks for mouse CNS to achieve full clarity.</li>
	</ol>
	</li>
</ol>
5. Refractive Index Matching and Immunostaining of Cleared CNS
NOTE: nRIMS (Nycodenz-based Refractive Index Matching Solution) consists of 0.8 g/mL Nycodenz powder dissolved in 30 mL base buffer (0.01% sodium azide and Tween-20 in 0.1 M PBS, pH 8.0). It is recommended that the solution is placed in a 37 &#176;C shaking incubator to allow for proper solvation of the powder.
<ol>
	<li>Incubate tissues in 0.1% Triton X-100 in 0.1 M PBS for 2 h, then block with 2% bovine serum albumin (BSA) in 0.1 M PBS for 6 h.</li>
	<li>Wash sections three times in PBST (0.1% Tween-20 in 0.1 M PBS) for 2 h. Stain sections with primary antibodies (in this case, an anti-rabbit PECAM-CD31 antibody, which stains blood vessels) for 2 days.</li>
	<li>Stain sections with secondary antibodies (in this case, a goat anti-rabbit IgG Cy3 fluorescent conjugate) in 2% BSA for 2 days.</li>
	<li>Wash labeled tissues three times in PBST for 2 h, and store in 15 mL nRIMS for 2 - 10 days.</li>
	<li>Before imaging, move labeled tissues to a small amount of nRIMS on 35- or 60-mm tissue culture dishes. Fix with silicone around the bottom edge of the dish.</li>
	<li>Add 1.5 - 2 mL of fresh nRIMS.</li>
</ol>
6. Image processing
<ol>
	<li>Acquire images of cleared tissues with tile scanning using a confocal laser-scanning microscopewith 10x magnification (see Table of Materials). For of Cy3-labeled tissue, use wavelengths of 550-600 nm.</li>
</ol>

<ol><li>Zhu, X., Xia, Y., Wang, X., Si, K., Gong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+brain+imaging%3A+A+powerful+tool+for+neuroscience%5BTitle%5D)+AND+%22Neurosci+Bull%22%5BJournal%5D)">Optical brain imaging: A powerful tool for neuroscience.</a> Neurosci Bull. 33 (1), 95-102 (2017).</li>
<li>Chung, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+molecular+interrogation+of+intact+biological+systems%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structural and molecular interrogation of intact biological systems.</a> Nature. 497 (7449), 332-337 (2013).</li>
<li>Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nat Protoc. 9 (7), 1682-1697 (2014).</li>
<li>Feng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CLARITY+reveals+dynamics+of+ovarian+follicular+architecture+and+vasculature+in+three-dimensions%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.</a> Sci Rep. 7, 44810(2017).</li>
<li>Lee, H., Park, J. H., Seo, I., Park, S. H., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+application+of+the+electrophoretic+tissue+clearing+technology%2C+CLARITY%2C+to+intact+solid+organs+including+brain%2C+pancreas%2C+liver%2C+kidney%2C+lung%2C+and+intestine%5BTitle%5D)+AND+%22BMC+Dev+Biol%22%5BJournal%5D)">Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine.</a> BMC Dev Biol. 14, 48(2014).</li>
<li>Jensen, K. H. R., Berg, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+and+perspectives+in+tissue+clearing+using+CLARITY%5BTitle%5D)+AND+%22J+Chem+Neuroanat%22%5BJournal%5D)">Advances and perspectives in tissue clearing using CLARITY.</a> J Chem Neuroanat. 86, 19-34 (2017).</li>
<li>Treweek, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-body+tissue+stabilization+and+selective+extractions+via+tissue-hydrogel+hybrids+for+high-resolution+intact+circuit+mapping+and+phenotyping%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.</a> Nat Protoc. 10 (11), 1860-1896 (2015).</li>
<li>Yang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158 (4), 945-958 (2014).</li>
<li>Neckel, P. H., Mattheus, U., Hirt, B., Just, L., Mack, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+tissue+clearing+%28PACT%29%3A+Technical+evaluation+and+new+perspectives+in+immunofluorescence%2C+histology%2C+and+ultrastructure%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Large-scale tissue clearing (PACT): Technical evaluation and new perspectives in immunofluorescence, histology, and ultrastructure.</a> Sci Rep. 6, 34331(2016).</li>
<li>Woo, J., Lee, M., Seo, J. M., Park, H. S., Cho, Y. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+the+optical+transparency+of+rodent+tissues+by+modified+PACT-based+passive+clearing%5BTitle%5D)+AND+%22Exp+Mol+Med%22%5BJournal%5D)">Optimization of the optical transparency of rodent tissues by modified PACT-based passive clearing.</a> Exp Mol Med. 48 (12), 274(2016).</li>
<li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole+animal+perfusion+fixation+for+rodents%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Whole animal perfusion fixation for rodents.</a> J Vis Exp. (65), (2012).</li>
<li>Roberts, D. G., Johnsonbaugh, H. B., Spence, R. D., MacKenzie-Graham, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+clearing+of+the+mouse+central+nervous+system+using+passive+CLARITY%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Optical clearing of the mouse central nervous system using passive CLARITY.</a> J Vis Exp. (112), (2016).</li>
<li>Ke, M. T., Fujimoto, S., Imai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SeeDB%3A+A+simple+and+morphology-preserving+optical+clearing+agent+for+neuronal+circuit+reconstruction%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">SeeDB: A simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.</a> Nat Neurosci. 16 (8), 1154-1161 (2013).</li>
<li>Choi, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+expression+of+the+receptor+for+advanced+glycation+end+products+in+neurons+and+astrocytes+in+a+triple+transgenic+mouse+model+of+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Exp+Mol+Med%22%5BJournal%5D)">Increased expression of the receptor for advanced glycation end products in neurons and astrocytes in a triple transgenic mouse model of Alzheimer's disease.</a> Exp Mol Med. 46, 75(2014).</li>
<li>Chang, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contralaterally+transplanted+human+embryonic+stem+cell-derived+neural+precursor+cells+%28ENStem-A%29+migrate+and+improve+brain+functions+in+stroke-damaged+rats%5BTitle%5D)+AND+%22Exp+Mol+Med%22%5BJournal%5D)">Contralaterally transplanted human embryonic stem cell-derived neural precursor cells (ENStem-A) migrate and improve brain functions in stroke-damaged rats.</a> Exp Mol Med. 45, 53(2013).</li>
<li>Kim, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+differential+plaque+depositions+in+the+brains+of+Tg2576+and+Tg-APPswe%2FPS1dE9+transgenic+mouse+models+of+Alzheimer+disease%5BTitle%5D)+AND+%22Exp+Mol+Med%22%5BJournal%5D)">Analysis of differential plaque depositions in the brains of Tg2576 and Tg-APPswe/PS1dE9 transgenic mouse models of Alzheimer disease.</a> Exp Mol Med. 44 (8), 492-502 (2012).</li>
<li>Kinameri, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prdm+proto-oncogene+transcription+factor+family+expression+and+interaction+with+the+Notch-Hes+pathway+in+mouse+neurogenesis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Prdm proto-oncogene transcription factor family expression and interaction with the Notch-Hes pathway in mouse neurogenesis.</a> PLoS One. 3 (12), e3859(2008).</li>
</ol>

The authors have nothing to disclose.

While the passive, non-electrophoretic extraction methods employed in PACT significantly improved the consistency achieved with previous tissue clearing methods such as CLARITY2,3,4,7,8, the technique still bears several shortcomings, the most pressing of which is the length of time required to achieve maximal tissue clarity12. In the c...

A fundamental objective of scientific and clinical inquiry involves attaining a complete understanding of organ structure and function; however, the exceedingly complex nature of mammalian organs often serves as a barrier to fully achieving this aim1. CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tisssue-hYdrogel)2,3,4, which involves building an acrylamide-based hydrogel hybrid from intact tissues, achieves optical clearance of a variety of organs, including the brain, liver, and spleen, while preserving their structural integrity5. CLARITY has thus enabled not only visualization but also the opportunity to finely dissect complex cellular networks and tissue morphologies without the need for sectioning.
In order to achieve tissue clearance, CLARITY employs electrophoretic methods to remove the lipid content of the sample at hand. While CLARITY has been noted for producing physically stable tissue-hydrogel hybrids, studies have shown that its use of electrophoretic tissue clearing (ETC) methods yields variable results in terms of tissue quality, including browning, epitope damage, and protein loss5,6. To address these issues, modified protocols such as PACT (PAssive Clarity Technique), which replaces the ETC treatment with a passive, ionic-detergent based delipidation technique, have been developed7,8,9. Despite achieving a greater consistency in results, however, PACT requires more time to obtain maximal clearance. Furthermore, none of these techniques have yet been applied to the whole CNS form, or in larger rodent models such as rats and guinea pigs.
The present study seeks to address these limitations by proposing novel methodologies, psPACT (process-separate PACT) and mPACT (modified PACT), for facilitating the fast clearance of the whole CNS and internal organs in both mouse and rat models10. Specifically, psPACT processes tissues in 4% acrylamide and 0.25% VA-044 in two separate steps during hydrogel formation; mPACT essentially involves the same steps, but supplements the SDS-based clearing solution with 0.5% &#945;-thioglycerol as a key reagent. Both techniques harness the endogenous systemic and cerebrospinal circulatory systems to significantly reduce the time needed to produce optical clearance. As a proof of principle, we demonstrate the use of confocal microscopy to analyze blood vessel patterns in the cleared tissues10.

<table><tbody><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Affymetrix, Inc.</td><td>75819</td><td>Clearing solution</td></tr><tr><td>Nycodenz</td><td>Axia-Shield</td><td>1002424</td><td>nRIMS solution</td></tr><tr><td>40% Acrylamide Solution</td><td>Bio Rad Laboratories, Inc.</td><td>161-0140</td><td>Polymerization (A4P0)</td></tr><tr><td>2,2&amp;acute;-Azobis[2-(2-imidazolin-2-yl)propane] Dihydrochloride</td><td>Wako Pure Chemical Industries, Ltd.</td><td>017-19362</td><td>Polymerization (VA-044)</td></tr><tr><td>1-Thioglycerol</td><td>Sigma-Aldrich</td><td>M1753-100ML</td><td>Clearing solution (mPACT)</td></tr><tr><td>Tween-20</td><td>Georgiachem</td><td>9005-64-5</td><td>nRIMS solution</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787-50ML</td><td>Immuno Staining</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Bovogen</td><td>BSA100</td><td>Immuno Staining</td></tr><tr><td>Heparin</td><td>Merck Millipore</td><td>375095</td><td>Perfusion (PBS)</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S2002-25G</td><td>nRIMS solution</td></tr><tr><td>PECAM-CD31 antibody</td><td>Santa Cruz Biotechnology Inc.</td><td>sc-28188</td><td>Immuno Staining</td></tr><tr><td>Goat anti-rabbit-IgG Cy3 fluorescent conjugate</td><td>Jackson ImmunoResearch Inc.</td><td>111-165-003</td><td>Immuno Staining</td></tr><tr><td>4% Paraformaldehyde</td><td>Tech &amp;amp; Innovation</td><td>BPP-9004</td><td>Perfusion, Polymerization</td></tr><tr><td>20X Phosphate Buffered Saline (pH 7.4)</td><td>Tech &amp;amp; Innovation</td><td>BPB-9121</td><td>Perfusion, Buffer</td></tr><tr><td>10 mL stripette</td><td>Coatar</td><td>4488</td><td>Solution transfer</td></tr><tr><td>50 mL tube</td><td>Falcon</td><td>352070</td><td>Clearing tube</td></tr><tr><td>35 mm Cell culture dish</td><td>SPL</td><td>20035</td><td>Imaging</td></tr><tr><td>Confocal dish</td><td>SPL</td><td>211350</td><td>Imaging</td></tr><tr><td>1 mL syringe</td><td>Korea vaccine Co., Ltd</td><td>26G 1/2</td><td>Anesthetize&amp;nbsp;</td></tr><tr><td>50 mL syringe</td><td>Korea vaccine Co., Ltd</td><td>21G1 1/4</td><td>Perfusion</td></tr><tr><td>Acrylamide</td><td>Sigma-Aldrich</td><td>A3553</td><td>Polymerization (A4P0)</td></tr><tr><td>Whatman 3MM paper</td><td>Sigma-Aldrich</td><td>Z270849</td><td>Blotting paper for gel removal</td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM780</td><td>Imaging</td></tr><tr><td>ZEN lite Software</td><td>Zeiss</td><td>ZEN 2012</td><td>Imaging</td></tr><tr><td>Peristaltic pump</td><td>Longerpump</td><td>BT100-1F</td><td>Perfusion</td></tr><tr><td>EasyGel</td><td>Lifecanvas Technologies</td><td>EasyGel</td><td>Tissue gel hybridization system</td></tr></tbody></table>

novel passive clearing methods for the rapid production of optical transparency in whole cns tissue

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Generation of a transparent model of the whole CNS using optimized passive clearing techniques
Optical clearance of mouse and rat whole CNS tissues was rapidly achieved using various passive clearing techniques (Figure 1). A schematic of tissue clearing over time is shown in Figure 2A. Unlike the original PACT method, psPACT (process-separate PACT) invo...

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

novel-passive-clearing-methods-for-rapid-production-optical

Research

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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.

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

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 and Imaging of Immunolabeled Kidney Tissue

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.

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

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 Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing.

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

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.

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

This protocol combines optical clearing and immunolabeling for full-volume confocal imaging of whole-mount retinal organoids. It preserves 3D structure, enabling detailed visualization of key neuronal pathways, improving the study of retinal maturation, spatial organization, and its potential applications in disease modeling and personalized medicine.

Whole-mount Retinal Organoid Visualization with Cellular Resolution

Optical clearing techniques, such as absorbing molecules solutions, reduce light scattering temporarily to enhance biological imaging and offer biocompatible, reversible in vivo imaging strategies for deeply embedded organs. This protocol presents detailed experimental procedures as well as advanced image processing to achieve high-quality, dynamic imaging for real-time dynamics in living animals.

Transient Optical Clearing Using Absorbing Molecules for Ex Vivo and In Vivo Imaging

Discover psPACT and mPACT methods for rapid optical clearing of whole rodent CNS tissue without electrophoretic damage. These techniques enable efficient microscopic visualization of vasculature for neuroscience research.

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

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