Name: visualization of replisome encounters with an antigen tagged blocking lesion
Uploaded: 2021-07-27T13:00:00
Description: Watch this Scientific Journal Video about Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion at JoVE.com

This research was supported, in part, by the Intramural Research Program of the NIH, National Institute on Aging, United States (Z01-AG000746&#8211;08). J.H. is supported by the National Natural Science Foundations of China (21708007 and 31871365).

1. Cell preparation
<ol>
	<li>Day 1
	<ol>
		<li>Pre-treat 35 mm glass-bottomed culture dishes with a cell adhesive solution.</li>
		<li>Plate cells in the pre-treated dishes one day before treatment. Cell should be actively dividing and 50&#8211;70% confluent on the day of the experiment. 
		NOTE: HeLa cells were used in this experiment with Dulbecco Modified Eagle Medium DMEM, supplemented with 10% fetal bovine serum, 1x penicillin /streptomycin. There is no restriction for adherent cell lines. However, non-adher...

<ol>
	<li>Rouet, P., Smih, F., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+double-strand+breaks+into+the+genome+of+mouse+cells+by+expression+of+a+rare-cutting+endonuclease.">Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.</a> Molecular and Cellular Biology. 14 (12), 8096-8106 (1994).</li><li>Wright, D. 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=Standardized+reagents+and+protocols+for+engineering+zinc+finger+nucleases+by+modular+assembly.">Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly.</a> Nature Protocols. 1 (3), 1637-1652 (2006).</li><li>Brinkman, E. 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=Kinetics+and+fidelity+of+the+repair+of+Cas9-induced+double-strand+DNA+breaks.">Kinetics and fidelity of the repair of Cas9-induced double-strand DNA breaks.</a> Molecular Cell. 70 (5), 801-813 (2018).</li><li>Vitor, A. C., Huertas, P., Legube, G., de Almeida, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+DNA+double-strand+break+repair:+An+ever-growing+toolbox.">Studying DNA double-strand break repair: An ever-growing toolbox.</a> Frontiers in Molecular Bioscience. 7, 24 (2020).</li><li>Galbiati, A., Beausejour, C., d'Adda di, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+single-cell+method+provides+direct+evidence+of+persistent+DNA+damage+in+senescent+cells+and+aged+mammalian+tissues.">A novel single-cell method provides direct evidence of persistent DNA damage in senescent cells and aged mammalian tissues.</a> Aging Cell. 16 (2), 422-427 (2017).</li><li>Vitelli, V., 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=Recent+Advancements+in+DNA+damage-transcription+crosstalk+and+high-resolution+mapping+of+DNA+breaks.">Recent Advancements in DNA damage-transcription crosstalk and high-resolution mapping of DNA breaks.</a> Annual Review of Genomics and Human Genetics. 18, 87-113 (2017).</li><li>Canela, 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=DNA+breaks+and+end+resection+measured+genome-wide+by+end+sequencing.">DNA breaks and end resection measured genome-wide by end sequencing.</a> Molecular Cell. 63 (5), 898-911 (2016).</li><li>Muniandy, P. A., Liu, J., Majumdar, A., Liu, S. T., Seidman, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+interstrand+crosslink+repair+in+mammalian+cells:+step+by+step.">DNA interstrand crosslink repair in mammalian cells: step by step.</a> Critical Reviews in Biochemistry and Molecular Biology. 45 (1), 23-49 (2010).</li><li>Nejad, M. I., 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=Interstrand+DNA+cross-links+derived+from+reaction+of+a+2-aminopurine+residue+with+an+abasic+site.">Interstrand DNA cross-links derived from reaction of a 2-aminopurine residue with an abasic site.</a> ACS Chemical Biology. 14 (7), 1481-1489 (2019).</li><li>Kottemann, M. C., Smogorzewska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fanconi+anaemia+and+the+repair+of+Watson+and+Crick+DNA+crosslinks.">Fanconi anaemia and the repair of Watson and Crick DNA crosslinks.</a> Nature. 493 (7432), 356-363 (2013).</li><li>Kaushal, S., Freudenreich, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+fork+stalling+and+DNA+structures+in+causing+chromosome+fragility.">The role of fork stalling and DNA structures in causing chromosome fragility.</a> Genes Chromosomes Cancer. 58 (5), 270-283 (2019).</li><li>Knipscheer, P., Raschle, M., Scharer, O. D., Walter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-coupled+DNA+interstrand+cross-link+repair+in+Xenopus+egg+extracts.">Replication-coupled DNA interstrand cross-link repair in Xenopus egg extracts.</a> Methods in Molecular Biology. 920, 221-243 (2012).</li><li>Klein, D. D., 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=XPF-ERCC1+acts+in+Unhooking+DNA+interstrand+crosslinks+in+cooperation+with+FANCD2+and+FANCP/SLX4.">XPF-ERCC1 acts in Unhooking DNA interstrand crosslinks in cooperation with FANCD2 and FANCP/SLX4.</a> Molecular Cell. 54 (3), 460-471 (2014).</li><li>Long, D. T., Raschle, M., Joukov, V., Walter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+RAD51-dependent+DNA+interstrand+cross-link+repair.">Mechanism of RAD51-dependent DNA interstrand cross-link repair.</a> Science. 333 (6038), 84-87 (2011).</li><li>Benedetto, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+psoralens.+An+historical+perspective.">The psoralens. An historical perspective.</a> Cutis. 20 (4), 469-471 (1977).</li><li>Huang, J., 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=The+DNA+translocase+FANCM/MHF+promotes+replication+traverse+of+DNA+interstrand+crosslinks.">The DNA translocase FANCM/MHF promotes replication traverse of DNA interstrand crosslinks.</a> Molecular Cell. 52 (3), 434-446 (2013).</li><li>Thazhathveetil, A. K., Liu, S. T., Indig, F. E., Seidman, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psoralen+conjugates+for+visualization+of+genomic+interstrand+cross-links+localized+by+laser+photoactivation.">Psoralen conjugates for visualization of genomic interstrand cross-links localized by laser photoactivation.</a> Bioconjugate Chemistry. 18 (2), 431-437 (2007).</li><li>O'Donnell, M. E., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ring-shaped+hexameric+helicases+that+function+at+DNA+replication+forks.">The ring-shaped hexameric helicases that function at DNA replication forks.</a> Nature Structural &amp; Molecular Biology. 25 (2), 122-130 (2018).</li><li>Koos, 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=Analysis+of+protein+interactions+in+situ+by+proximity+ligation+assays.">Analysis of protein interactions in situ by proximity ligation assays.</a> Current Topics in Microbiology and Immunology. 377, 111-126 (2014).</li><li>Huang, J., 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=Remodeling+of+Interstrand+Crosslink+Proximal+Replisomes+Is+Dependent+on+ATR,+FANCM,+and+FANCD2.">Remodeling of Interstrand Crosslink Proximal Replisomes Is Dependent on ATR, FANCM, and FANCD2.</a> Cell Reports. 27 (6), 1794-1808 (2019).</li><li>Huang, J., 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+molecule+analysis+of+laser+localized+psoralen+adducts.">Single molecule analysis of laser localized psoralen adducts.</a> Journal of Visualized Experiments. (122), e55541 (2017).</li><li>Saldivar, J. C., Cortez, D., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+kinase+ATR:+ensuring+faithful+duplication+of+a+challenging+genome.">The essential kinase ATR: ensuring faithful duplication of a challenging genome.</a> Nature Reviews Molecular Cell Biology. 18 (10), 622-636 (2017).</li><li>Cortez, D., Glick, G., Elledge, 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=Minichromosome+maintenance+proteins+are+direct+targets+of+the+ATM+and+ATR+checkpoint+kinases.">Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases.</a> Proceedings of the National Academy of Sciences. 101 (27), 10078-10083 (2004).</li><li>Ersoy, I., Bunyak, F., Chagin, V., Cardoso, M. C., Palaniappan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmentation+and+classification+of+cell+cycle+phases+in+fluorescence+imaging.">Segmentation and classification of cell cycle phases in fluorescence imaging.</a> Medical Image Computing and Computer-Assisted. 12, 617-624 (2009).</li><li>Zhao, J., Dynlacht, B., Imai, T., Hori, T., Harlow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+NPAT,+a+novel+substrate+of+cyclin+E-CDK2,+promotes+S-phase+entry.">Expression of NPAT, a novel substrate of cyclin E-CDK2, promotes S-phase entry.</a> Genes &amp; Development. 12 (4), 456-461 (1998).</li></ol>

Although the PLA is a very powerful technique, there are technical concerns that must be solved in order to obtain clear and reproducible results. The antibodies must be of high affinity and specificity. Furthermore, it is important to reduce the non-specific background signals as much as possible. We have found that membranes and cellular debris contribute to the background, and we have removed them as much as possible. The washes with detergent containing buffers prior to fixing, and the wash with methanol after fixing...

An erratum was issued for: <a href="https://www.jove.com/t/6517">Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion</a>.
The Authors section was updated from:
Jing Zhang*1 
Jing Huang*2 
Ryan C. James3 
Julia Gichimu1 
Manikandan Paramasivam4 
Durga Pokharel5 
Himabindu Gali6 
Marina A. Bellani1 
Michael M Seidman1 
1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health 
2Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University 
3Department of Molecular Biology and Genetics, Cornell University 
4Department of Cellular and Molecular Medicine, University of Copenhagen 
5Horizon Discovery 
6Boston University School of Medicine 
* These authors contributed equally
to:
Jing Zhang*1 
Jing Huang*2 
Ishani Majumdar1 
Ryan C. James3 
Julia Gichimu1 
Manikandan Paramasivam4 
Durga Pokharel5 
Himabindu Gali6 
Marina A. Bellani1 
Michael M Seidman1 
1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health 
2Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University 
3Department of Molecular Biology and Genetics, Cornell University 
4Department of Cellular and Molecular Medicine, University of Copenhagen 
5Horizon Discovery 
6Boston University School of Medicine 
* These authors contributed equally

An erratum was issued for: <a href="https://www.jove.com/t/6517">Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion</a>. The Authors section was updated.

Erratum: Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion

The cellular response to double strand breaks is well documented owing to a succession of increasingly powerful methods for directing breaks to specific genomic sites1,2,3. The certainty of location enables unambiguous characterization of proteins and other factors that accumulate at the site and participate in the DNA Damage Response (DDR) thereby driving the Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) pathways that repair breaks. Of course, many breaks are introduced by agents such as radiation and chemical species that do not attack specific sequences4. However, for these there are procedures available that can convert the ends to structures amenable for tagging and localization5,6. Breaks are also introduced by biological processes, such as immunoglobulin rearrangement, and recent technology permits their localization, as well7. The relationship between responding factors and those sites can then be determined.
Breaks also appear as an indirect consequence of adducts formed by compounds that are not inherent breakers but disrupt DNA transactions such as transcription and replication. They may be formed as a feature of the cellular response to these obstructions, perhaps during repair or because they provoke a structure that is vulnerable to nuclease attack. Typically, the physical relationship between the adduct, the break, and the association with responding factors is inferential. For example, ICLs are formed by chemotherapeutics such as cisplatin and Mitomycin C8 and as a reaction product of abasic sites9. ICLs are well known as potent blocks to replication forks10, thereby stalling forks which can be cleaved by nucleases11. The covalent linkage between strands is often relieved by pathways that have obligate breaks as intermediates12,13, necessitating homologous recombination to rebuild the replication fork14. In most experiments the investigator follows the response of factors of interest to the breaks which are formed downstream of the collision of a replication fork with an ICL. However, because there has been no technology for the localization of a provocative lesion, the proximity of the replisome, and its component parts, to the ICL can only be assumed.
We have developed a strategy to enable the analysis of protein associations with non-sequence specific covalent adducts, illustrated here by ICLs. In our system these are introduced by psoralen, a photoactive natural product used for thousands of years as a therapeutic for skin disorders15. Our approach is based on two important features of psoralens. The first is their high frequency of crosslink formation, which can exceed 90% of adducts, in contrast to the less than 10% formed by popular compounds such as cisplatin or Mitomycin C8,16. The second is the accessibility of the compound to conjugation without loss of crosslinking capacity. We have covalently linked trimethyl psoralen to Digoxigenin (Dig), a long established immunotag. This enables detection of the psoralen adducts in genomic DNA by immunostaining of the Dig tag, and visualization by conventional immunofluorescence17.
This reagent was applied, in our previous work, to the analysis of replication fork encounters with ICLs using a DNA fiber-based assay16. In that work we found that replication could continue past an intact ICL. This was dependent on the ATR kinase, which is activated by replication stress. The replication restart was unexpected given the structure of the CMG replicative helicase. This consists of the MCM hetero-hexamer (M) that forms an offset gapped ring around the template strand for leading strand synthesis which is locked by the proteins of the GINS complex (G, consisting of PSF1, 2, 3, and SLD5) and CDC45 (C)18. The proposal that replication could restart on the side of the ICL distal to the side of the replisome collision argued for a change in the structure of the replisome. To address the question of which components were in the replisome at the time of the encounter with an ICL we developed the approach described here. We exploited the Dig tag as a partner in Proximity Ligation Assays (PLA)19 to interrogate the close association of the ICL with proteins of the replisome20.

<table>
	<tbody>
		<tr>
			<td>Alexa Fluor 568, Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody</td>
			<td>Invitrogen</td>
			<td>A-10011</td>
			<td>1 in 1000</td>
		</tr>
		<tr>
			<td>35 mm plates with glass 1.5 coverslip</td>
			<td>MatTek</td>
			<td>P35-1.5-14-C</td>
			<td>Glass Bottom Microwell Dishes 35mm Petri Dish Microwell</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488,Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody</td>
			<td>Invitrogen</td>
			<td>A-10001</td>
			<td>1 in 1000</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>SeraCare</td>
			<td>1900-0012</td>
			<td>Blocking solution, reagents need to be stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>CDC45 antibody (rabbit)</td>
			<td>Abcam</td>
			<td>ab126762</td>
			<td>1 in 200</td>
		</tr>
		<tr>
			<td>Cell adhesive</td>
			<td>Life Science</td>
			<td>354240</td>
			<td>for cell-TAK solution</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikkon</td>
			<td>Nikon TE2000 spinning disk microscope</td>
			<td>equiped with Volocity software</td>
		</tr>
		<tr>
			<td>Digoxigenin (Dig) antibody (mouse)</td>
			<td>Abcam</td>
			<td>ab420</td>
			<td>1 in 200</td>
		</tr>
		<tr>
			<td>Dig-TMP</td>
			<td>synthesized in the Seidman Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink Amplification reagents (5&#215;)</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82010</td>
			<td>reagents need to be stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Duolink in situ detection reagents</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92007</td>
			<td>reagents need to be stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Duolink in situ oligonucleotide PLA probe MINUS</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92004</td>
			<td>anti-mouse MINUS, reagents need to be stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Duolink in situ oligonucleotide PLA probe PLUS</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92002</td>
			<td>anti-rabbit PLUS, reagents need to be stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Duolink in situ wash buffer A</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82046</td>
			<td>Duolink Wash Buffers, reagents need to be stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Duolink in situ wash buffer B</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82048</td>
			<td>Duolink Wash Buffers, reagents need to be stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>epifluorescent microscope</td>
			<td>Zeiss</td>
			<td>Axiovert 200M microscope</td>
			<td>Equipped with the Axio Vision software packages (Zeiss, Germany)</td>
		</tr>
		<tr>
			<td>Formaldehyde 16%</td>
			<td>Fisher Scientific</td>
			<td>PI28906</td>
			<td>for fix solution</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Thermo</td>
			<td>31873</td>
			<td>Blocking solution, reagents need to be stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>open source</td>
			<td>Cell profiler</td>
			<td>works for analysis of single plane images</td>
		</tr>
		<tr>
			<td>Image analysis software-license required</td>
			<td>Bitplane</td>
			<td>Imaris</td>
			<td>Cell Biology module needed. Can quantify PLA dots/nuclei in image stacks (3D) and do 3D reconstructions</td>
		</tr>
		<tr>
			<td>Ligase (1 unit/&#956;l)</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82029</td>
			<td>reagents need to be stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Ligation reagent (5&#215;)</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82009</td>
			<td>reagents need to be stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>MCM2 antibody (rabbit)</td>
			<td>Abcam</td>
			<td>ab4461</td>
			<td>1 in 200</td>
		</tr>
		<tr>
			<td>MCM5 antibody (rabbit monoclonal)</td>
			<td>Abcam</td>
			<td>Ab75975</td>
			<td>1 in 1000</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Lab ALLEY</td>
			<td>A2076</td>
			<td>pre-cold at -20&#176;C before use</td>
		</tr>
		<tr>
			<td>phosphoMCM2S108 antibody (rabbit)</td>
			<td>Abcam</td>
			<td>ab109271</td>
			<td>1 in 200</td>
		</tr>
		<tr>
			<td>Polymerase (10 unit/&#956;l)</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82030</td>
			<td>reagents need to be stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Prolong gold mounting media with DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>PSF1 antibody (rabbit)</td>
			<td>Abcam</td>
			<td>ab181112</td>
			<td>1 in 200</td>
		</tr>
		<tr>
			<td>RNAse A 100 mg/ml</td>
			<td>Qiagen</td>
			<td>19101</td>
			<td>reagents need to be stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Statistical analysis and data visualization software</td>
			<td>open source</td>
			<td>R studio</td>
			<td>ggplot2 package for generation of dot plot and box plots</td>
		</tr>
		<tr>
			<td>Statistical analysis and data visualization software-license required</td>
			<td>Systat Software</td>
			<td>Sigmaplot V13</td>
			<td></td>
		</tr>
		<tr>
			<td>TMP (trioxalen)</td>
			<td>Sigma-Aldrich</td>
			<td>T6137_1G</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787_250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416_100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>UV box</td>
			<td>Southern New England Ultraviolet</td>
			<td></td>
			<td>Discontinued. See Opsytec UV test chamber as a possible replacement</td>
		</tr>
		<tr>
			<td>UV test Chamber</td>
			<td>Opsytec</td>
			<td>UV TEST CHAMBER BS-04</td>
			<td></td>
		</tr>
		<tr>
			<td>VE-821</td>
			<td>Selleckchem</td>
			<td>S8007</td>
			<td>final concentrtion is 1&#181;M</td>
		</tr>
	</tbody>
</table>

visualization of replisome encounters with an antigen tagged blocking lesion

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In part, this reflects the availability of methods for the identification of break sites, and characterization of factors recruited to DSBs at those sequences. However, DSBs also appear as intermediates during the processing of DNA adducts formed by compounds that do not directly cause breaks, and do not react at specific sequence sites. Consequently, for most of these agents, technologies that permit the analysis of binding interactions with response factors and repair proteins are unknown. For example, DNA interstrand crosslinks (ICLs) can provoke breaks following replication fork encounters. Although formed by drugs widely used as cancer chemotherapeutics, there has been no methodology for monitoring their interactions with replication proteins.
Here, we describe our strategy for following the cellular response to fork collisions with these challenging adducts. We linked a steroid antigen to psoralen, which forms photoactivation dependent ICLs in nuclei of living cells. The ICLs were visualized by immunofluorescence against the antigen tag. The tag can also be a partner in the Proximity Ligation Assay (PLA) which reports the close association of two antigens. The PLA was exploited to distinguish proteins that were closely associated with the tagged ICLs from those that were not. It was possible to define replisome proteins that were retained after encounters with ICLs and identify others that were lost. This approach is applicable to any structure or DNA adduct that can be detected immunologically.

PLA of Dig-TMP with replisome proteins 
The structure of the Dig-TMP is shown in Figure 1. The details of the synthesis, in which trimethyl psoralen was conjugated through a glycol linker to digoxigenin, have been discussed previously17,21. Incubation of cells with the compound followed by exposure to 365 nm light (UVA) photoactivates the compound and drives the crosslinking reaction. Slightly more than 90% of a...

Watch this Scientific Journal Video about Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion at JoVE.com

Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion

While replication fork collisions with DNA adducts can induce double strand breaks, less is known about the interaction between replisomes and blocking lesions. We have employed the proximity ligation assay to visualize these encounters and to characterize the consequences for replisome composition.

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In ...

This technique facilitates the visualization of replisome antigen-tagged DNA adduct encounters and can be extended to the interactions of any protein with a DNA structure or lesion amenable to immunodetection. This method can be used to assess interaction frequencies within individual cells, rather than across a population, and allows the visualization of cell cycle-dependent interactions in situ without damaging cells. Demonstrating the procedure will be Ishani Majumdar, a post-doc from the Seidman Laboratory.On the day before the experiment, seed 1.5 times 10 to the fifth cells in two milliliters of medium onto 35-millimeter glass bottom culture dishes pre-treated with cell adhesive solution. The next day, replace the supernatant in each dish with 37 degrees Celsius Five micromolar Dig TMP-supplemented medium to the 50 to 70%confluent cell cultures and return the plates to the cell culture incubator for 30 minutes to allow the Dig TMP to equilibrate. While the cells are incubating, prewarm a UV box to 37 degrees Celsius.At the end of the equilibration, transfer the plates to the pre-warmed box to expose the cells to a five-minute three-joule-per-square-centimeter UVA light dose and replace the supernatants with two milliliter of fresh pre-warmed culture medium before returning the plates to the cell culture incubator for one hour. At the end of the incubation, gently wash the cultures with PBS and fix the cells with one milliliter of 0.1%formaldehyde in PBS for five minutes at room temperature. At the end of the incubation, wash the cells one time with PBS before treating the cells two times with 80 microliters of cytoskeleton extraction buffer supplemented with RNase for five minutes at room temperature per treatment to remove the cytoplasm.After the second incubation, wash the cells with two milliliter of PBS three times before fixing the cells with one milliliter of 4%formaldehyde in PBS for 10 minutes at room temperature. After the second fixation, wash the cells three times with PBS as demonstrated, followed by treatment with one milliliter of cold 100%methanol for 20 minutes at minus 20 degrees Celsius. At the end of the incubation, wash the cells three times in two milliliters of PBS, followed by a 10 minute incubation in 80 microliters of five millimolar Triton X-100 at four degrees Celsius.At the end of the incubation, treat the cells with 100 microliters of five millimolar EDTA in PBS, supplemented with one microliter of 100-milligram-per-milliliter of RNase A for 30 minutes at 37 degrees Celsius. At the end of the incubation, wash the cells three times in PBS before storing them in 5%bovine serum albumin and 10%donkey serum in PBS in a humid chamber overnight at four degrees Celsius. To perform a proximity ligation assay, add 40 microliters of the primary antibody solution of interest to the center of each plate and incubate the plates in the humid chamber for one hour at 37 degrees Celsius.At the end of the incubation, wash the cells three times with two milliliters of PBST before adding 40 microliters of freshly prepared PLA probe solution to each dish for a one hour incubation in the cell culture incubator. At the end of the incubation, wash the cells with three 10-minute washes in two milliliters of buffer A on a tilting platform at room temperature. After the last wash, add 40 microliters of freshly prepared ligation mix to each plate for a 30-minute incubation at 37 degrees Celsius, followed by three two-minute washes in buffer A on the tilting platform.After the last wash, add 40 microliters of freshly prepared amplification solution to each plate for a 100-minute incubation at 37 degrees Celsius in a humid chamber. At the end of the incubation, wash the cells six times with fresh buffer B for 10 minutes per wash on the tilting platform. After the last buffer B wash, wash the cells one time with 0.01x buffer B for one minute at room temperature before labeling with the appropriate secondary antibodies in the humid chamber for 30 minutes at 37 degrees Celsius.At the end of the incubation, wash the samples with three 10-minute washes on a tilting platform in PBST at room temperature before mounting them in mounting medium supplemented with DAPI. within four days of mounting, image at least 100 observations per sample or condition cells on an epifluorescence or confocal microscope, using the same exposure settings for both experimental and control samples. The Dig tag can be visualized by immunofluorescence to reveal the presence of interstrand crosslinks throughout the nucleus.To visualize the interaction of replisomes with interstrand crosslinks, PLA can be applied to visualize the frequency of association of MCM5 and the Dig tag, one hour after interstrand crosslink introduction. As replication stress activates ATR kinase, the PLA between pMCM two serine 108 and the Dig tag is also positive. The PLA results from individual nuclei can be presented in a three-dimensional reconstruction allowing the visualization of replisome interstrand crosslink encounters throughout the nucleus.Replication Fork analysis indicates that interstrand crosslink encounters demonstrate an unexpected replication restart phenomenon, as the proteins of the GINS complex fail to exhibit PLA signal with the interstrand crosslinks. In contrast, the assay with CD 45 is positive, indicating that the other locking protein is retained. When cells are incubated with an ATR inhibitor, the restart is completely suppressed and the GINS Dig PLAs strongly positive.CSK+R treatment is essential for reducing background signal. Be sure to coat the plates with a cell adhesive and to prefix with 0.1%FA or the cells will peel off the plate.

visualization-replisome-encounters-with-an-antigen-tagged-blocking

Research

JoVE Journal

Biology

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for isolation of protein complexes under physiological conditions. Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, have the advantage of higher affinity for Strep-Tactin compared to those containing only a single Strep-tag, thus allowing more efficient protein purification. However, this advantage is offset by the fact that elution of twin-Strep-tagged proteins with biotin may be incomplete, leading to low protein recovery. The recovery can be dramatically improved by using denaturing elution with sodium dodecyl sulfate (SDS), but this leads to sample contamination with Strep-Tactin released from the resin, making the assay incompatible with downstream proteomic analysis. To overcome this limitation, we have developed a method whereby resin-coupled tetramer of Strep-Tactin is first stabilized by covalent cross-linking with Bis(sulfosuccinimidyl) suberate (BS3) and the resulting cross-linked resin is then used to purify target protein complexes in a single batch purification step. Efficient elution with SDS ensures good protein recovery, while the absence of contaminating Strep-Tactin allows downstream protein analysis by mass spectrometry. As a proof of concept, we describe here a protocol for purification of SIII-tagged viral protein VPg-Pro from nuclei of virus-infected N. benthamiana plants using the Strep-Tactin polymethacrylate resin cross-linked with BS3. The same protocol can be used to purify any twin-Strep-tagged protein of interest and characterize its physiological binding partners.

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

A method is described for efficient purification of twin-Strep-tagged fusion proteins and their specific complexes on modified streptavidin (Strep-Tactin) resin covalently cross-linked with Bis(sulfosuccinimidyl) suberate (BS3). The method has the advantages of fast speed, good target protein recovery and high purity, and is compatible with subsequent analysis by mass spectrometry.

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...