This work was supported by the Faculty Start-up fund from the Carnegie Mellon University (YZ) and NIH Director&#8217;s New Innovator Award (DP2 OD025926-01 to YZ).

1. Preparation of Stock Reagents and Solutions
<ol>
	<li>Prepare gelling solution components. 
	NOTE: Solution concentrations are given in g/mL (w/v percent).
	<ol>
		<li>Make the following stock solutions: 38% (w/v) sodium acrylate (SA), 50% (w/v) acrylamide (AA), 2% (w/v) N,N&#8242;-methylenebisacrylamide (Bis), and 29.2% (w/v) sodium chloride (NaCl). Dissolve the compounds in doubly deionized water (ddH2O). Use the amounts in Table 1 as a reference; prepared solutions can be scaled up or down in volume as needed. For example, to make 10 mL of a 38% (w/v) SA solution, add 1.9 g SA to a graduated 10 mL cylinder and add ddH2O to a volume of 5 mL.</li>
		<li>Prepare 9.4 mL of monomer solution at a 1.06x concentration as shown in Table 1. 
		NOTE: This will result in a 1x concentration after addition of the initiator, accelerator, and inhibitor. The monomer stock can be stored at 4 &#176;C for up to 3 months, or at -20 &#176;C for long-term storage.</li>
		<li>Prepare the following stock solutions separately in ddH2O: 0.5% (w/v) of the inhibitor 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4HT), which inhibits gelation to enable diffusion of the gelling solution into tissues, 10% (v/v) of the initiator tetramethylethylenediamine (TEMED), which accelerates radical generation by ammonium persulfate (APS), and 10% (w/v) APS which initiates the gelling process. 
		NOTE: Stock solutions of 4HT and TEMED can be prepared in 1 mL aliquots and stored at -20 &#176;C for at least 6 months. APS has been found to lose efficacy after long-term storage and is best prepared in small quantities (&#60;0.1 mL) immediately before gelling.</li>
	</ol>
	</li>
	<li>Prepare digestion buffer (50 mM Tris pH 8.0, 25 mM EDTA, 0.5% [w/v] nonionic surfactant, 0.8 M NaCl) by combining 25 mL of 1 M Tris pH 8 (3.03 g of Tris base in 25 mL of ddH2O), 25 mL of EDTA (0.5 M pH 8), 2.25 g of nonionic surfactant, and 23.38 g of NaCl. Add ddH2O for a total volume of 500 mL. 
	NOTE: The solution can be scaled up or down as needed and be stored in at 4 &#176;C. Proteinase K (ProK) will be added immediately before the digestion step.</li>
	<li>Prepare 20 mM sodium citrate solution by combining 2.941 g of sodium citrate tribasic dihydrate with 500 mL of ddH2O and adjusting pH to 8.0 at room temperature (RT). Scale the volume of stock as needed.</li>
	<li>Prepare a stock solution of 6-((acryloyl)amino)hexanoic acid, succinimidyl ester (acryloyl-X, SE; AcX), the anchoring compound. Dissolve AcX in 500 &#181;L of anhydrous dimethyl sulfoxide (DMSO) for a final concentration of 10 mg/mL. 
	NOTE: The solution can be stored in a desiccated environment at -20 &#176;C in 20 &#181;L aliquots.</li>
	<li>If not using commercially available buffers for immunostaining, prepare blocking buffer. Use a blocking buffer of 5% (v/v) normal animal serum and 0.1% (w/v) nonionic surfactant in 1x phosphate-buffered saline (PBS) and select the serum based on the host animal of the secondary antibodies. For example, to prepare 500 mL blocking buffer for antibodies raised in goat, combine 25 mL of goat serum, 0.45 g of nonionic surfactant, and 1x PBS to a volume of 500 mL.</li>
</ol>
2. Preparation of Archived and Freshly Prepared Clinical Tissue Slides for ExPath
<ol>
	<li>Convert the tissue into an ExPath compatible format. Choose one of the four following steps (2.1.1&#8722;2.1.4) based on how the specimen was prepared: FFPE slides, stained FFPE slides, or unfixed or fixed frozen tissue slides in optimum cutting temperature (OCT) solution. 
	NOTE: These are based on standard recovery steps for pathology samples and are not specific to the ExPath protocol.
	<ol>
		<li>FFPE clinical samples
		<ol>
			<li>Prepare 30 mL of 95% ethanol, 70% ethanol, and 50% ethanol. Measure out 30 mL of xylene, 100% ethanol, and ddH2O.</li>
			<li>Place the slide with the sample in a 50 mL conical using forceps and add 15 mL of xylene. Cap the tube and place it horizontally on an orbital shaker at approximately 60 rpm and incubate at RT for 3 min for each solution. Repeat with the remaining 15 mL xylene.</li>
			<li>Repeat step 2.1.1.2 with 100% ethanol, 95% ethanol, 70% ethanol, 50% ethanol, and ddH2O in place of xylene.</li>
		</ol>
		</li>
		<li>Stained and mounted permanent slides
		<ol>
			<li>Place the slide in a 100 mm Petri dish and cover with xylene. Carefully remove the coverslip using a razor blade. If the coverslip is not easily removed, return the slide to the xylene until the coverslip loosens.</li>
			<li>Process using the steps for FFPE samples (steps 2.1.1.1&#8722;2.1.1.3). 
			NOTE: In the case of H&#38;E stained slides, the stains are eliminated during the expansion process.</li>
		</ol>
		</li>
		<li>Unfixed frozen tissue slides in OCT solution
		<ol>
			<li>Fix the tissue in acetone at -20 &#176;C for 10 min.</li>
			<li>Wash the samples with 1x PBS solution 3 times for 10 min each at RT.</li>
		</ol>
		</li>
		<li>Previously fixed, frozen clinical tissue slides
		<ol>
			<li>Incubate the slides for 2 min at RT to melt the OCT solution.</li>
			<li>Wash the sample with 1x PBS solution 3 times for 5 min each at RT.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform heat treatment for antigen retrieval on all samples after format conversion.
	<ol>
		<li>Add 20 mM citrate solution (pH 8 at RT) in a heat resistant container, such as a slide staining jar. 
		NOTE: There should be enough solution to cover the tissue mounted on the slide (50 mL for a standard slide staining jar).</li>
		<li>Heat the citrate solution to 100 &#176;C in the microwave and place the slide in the solution. Immediately transfer the container to an incubation chamber and incubate at 60 &#176;C for 30 min. 
		NOTE: The protocol can be paused here. Slides can be placed in Petri dishes and covered in 1x PBS and stored at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Stain the sample using standard immunofluorescence (IF)/immunohistochemistry (IHC) staining protocols. 
	NOTE: Specific primary and secondary antibody concentrations and staining durations are dependent on the concentrations suggested by the manufacturer or by optimization for the specific experiment.
	<ol>
		<li>Use a hydrophobic pen to draw a boundary around the tissue section(s) on the slide to minimize the volume of solution needed to cover the tissue. Place the slide in a dish large enough to fit the slide. For a standard 3-inch slide, use a 100 mm Petri dish. 
		NOTE: The hydrophobic pen does not interfere with the polymerization of the sample nor the digestion process.</li>
		<li>Incubate the tissue with blocking buffer for 1 h at 37 &#176;C, 2 h at RT, or 4 &#176;C overnight to reduce nonspecific binding.</li>
		<li>Dilute primary antibodies to the desired concentration in the appropriate amount of prepared blocking buffer (or other preferred staining buffer). Incubate the tissues with the primary antibody solution for at least 3 h at RT or 37 &#176;C, or overnight at 4 &#176;C. 
		NOTE: Samples should be placed in a humidified container (such as a Petri dish with a damp wipe) to prevent the tissue from drying out. Typically, antibodies have been diluted to 1:100&#8722;1:500 in 200&#8722;500 &#181;L of buffer, depending on the tissue size and antibody used.</li>
		<li>Wash the tissue with prepared blocking buffer (or other preferred washing buffer) 3 times for 10 min at RT.</li>
		<li>Dilute secondary antibodies (and 300 nM 4&#8242;,6-diamidino-2-phenylindole [DAPI] if desired), in prepared blocking buffer (or other preferred staining buffer) to a concentration of approximately 10 &#181;g/mL. Incubate the tissue in the secondary antibody solution for at least 1 h at RT or 37 &#176;C. 
		NOTE: Timing may be adjusted depending on the antibodies used and the thickness of the tissue. Secondary antibodies containing cyanine dyes (Cy3, Cy5, Alexa 647) are not compatible with the ExM protocol when applied pre-polymerization. Suggested dyes include Alexa 488 (green), Alexa 546 (orange/red), and Atto 647N or CF633 (far-red). DAPI must be reapplied after expansion, as it is washed away during the expansion process.</li>
		<li>Wash the tissue with prepared blocking buffer (or other preferred washing buffer) 3 times for 10 min each at RT. 
		NOTE: The protocol can be paused here. Slides can be placed in Petri dishes and covered in 1x PBS and stored at 4 &#176;C.</li>
		<li>Perform fluorescent imaging using a conventional wide-field microscope, confocal microscope, or other imaging system of choice. 
		NOTE: This step is required to determine biological length using the expansion factor by comparing pre- and post-expansion images. To facilitate post-expansion imaging, easily identifiable regions of interest should be selected and images at both low and high magnification should be collected.</li>
	</ol>
	</li>
</ol>
3. In Situ Polymerization of Specimens
<ol>
	<li>Incubate the specimen in anchoring solution.
	<ol>
		<li>Prepare the anchoring solution (typically 250 &#181;L is enough to cover the tissue section) by diluting the AcX stock solution in 1x PBS to a concentration of 0.03 mg/mL for samples fixed with non-aldehyde fixatives or 0.1 mg/mL for samples fixed with aldehyde fixatives, which have fewer free amines available to react with AcX.</li>
		<li>Place the slide in a 100 mm Petri dish and pipette the anchoring solution over the tissue. Incubate for at least 3 h at RT or overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Incubate the samples in gelling solution.
	<ol>
		<li>Prepare at least 100-fold excess volume of gelling solution. Per 200 &#181;L, combine the following, in order: 188 &#181;L of monomer solution, 4 &#181;L of 0.5% 4HT stock solution (1:50 dilution, final concentration: 0.01%), 4 &#181;L of 10% TEMED stock solution (1:50 dilution, final concentration 0.2%), and 4 &#181;L of 10% APS stock solution (1:50 dilution, final concentration 0.2%). 
		NOTE: Gelling solution should be made immediately before use. The solution should be kept at 4 &#176;C and the APS solution should be added last, to prevent premature gelling.</li>
		<li>Remove excess solution from the tissue section and place the slide in a 100 mm Petri dish. Add fresh, cold gelling solution to the sample and incubate the mixture on the tissue for 30 min at 4 &#176;C, to allow diffusion of solution into the tissue.</li>
	</ol>
	</li>
	<li>Construct a chamber on the slide around the sample (Figure 2A) without disturbing the gelling solution.
	<ol>
		<li>Make spacers for the gelling chamber by thinly cutting pieces of cover glass using a diamond knife. 
		NOTE: To facilitate imaging post expansion, the spacers should be close in thickness to the tissue specimen to reduce the amount of blank gel above the tissue. Number 1.5 glass can be used for standard clinical samples (5&#8722;10 &#181;m). Cover glass pieces can be stacked for thicker samples.</li>
		<li>Secure the spacers on either side of the tissue using droplets of water (~10 &#181;L).</li>
		<li>Carefully place a cover glass lid over the slide, making sure to avoid trapping air bubbles over the tissue (Figure 2B).</li>
	</ol>
	</li>
	<li>Incubate the sample at 37 &#176;C in a humidified environment (such as a closed Petri dish with a damp wipe) for 2 h. 
	NOTE: The protocol can be paused here. The slide chamber can be stored inside a sealed Petri dish at 4 &#176;C.</li>
</ol>
4. Sample Digestion
<ol>
	<li>Remove the lid of the gelling chamber by gently sliding a razor blade under the coverslip and slowly lifting the coverslip off the gel surface. Trim the blank gel around the tissue to minimize volume. Cut the gel asymmetrically to track the orientation of the gel after homogenization, since the sample will become transparent.
	<ol>
		<li>Dilute ProK by 1:200 in digestion buffer (final concentration 4 U/mL) before use. Prepare enough solution to completely submerge the gel; a single well of a four-well plastic cell culture plate requires at least 3 mL per well.</li>
		<li>Incubate the sample in a closed container containing the digestion buffer for 3 h at 60 &#176;C. If the sample does not detach from the slide during digestion, use a razor blade to gently remove the sample. 
		NOTE: The specimen should be completely submerged in digestion buffer to prevent the sample from drying out and placed in a covered container (small slide box, plastic well, Petri dish, etc.) that can be sealed with film.</li>
	</ol>
	</li>
</ol>
5. Sample Expansion and Imaging
<ol>
	<li>Use a soft paint brush to transfer the specimen into 1x PBS in&#160;a container compatible with the desired imaging system and large enough to accommodate the fully expanded gel. Make sure that the tissue is placed with the sample-side down if imaging on an inverted system or up if imaging on an upright system to minimize the distance from the imaging objective to the sample. Flip the gel using a soft paint brush if needed. 
	NOTE: Side-illumination from an LED can be used to make them visible in liquid. A standard 6-well plate can accommodate samples that have a pre-expanded diameter less than 0.6 cm. A glass bottom well plate should be used for imaging on an inverted system.</li>
	<li>Wash the samples in 1x PBS at RT for 10 min. If desired, re-stain the sample with 300 nM DAPI as the digestion process washes away the DAPI stain. Remove PBS and stain with 300 nM DAPI diluted in 1x PBS for 20 min at RT, followed by a 10 min wash with 1x PBS at RT. 
	NOTE: The samples can be covered with 1x PBS and stored at 4 &#176;C before proceeding to the next step.</li>
	<li>To expand the samples, replace the PBS and wash with an excess volume of ddH2O (at least 10x the final gel volume) 3&#8722;5 times for 10 min each, at RT. 
	NOTE: After the 3rd or 4th wash, the specimen&#8217;s expansion should begin to plateau. For storage, to prevent bacterial growth, the ddH2O can be supplemented with 0.002%&#8722;0.01% sodium azide (NaN3). In this case, the final expansion factor is reversibly reduced by 10%.</li>
	<li>Perform fluorescence imaging using a conventional wide-field microscope, confocal microscope, or other imaging system of choice. 
	NOTE: To prevent gels from drifting, excess liquid can be removed from the well. Gels can also be immobilized with 1.5&#8722;2% low-melt agarose. Prepare 1.5&#8722;2%(w/v) low-melt agarose in water in a container 2&#8722;4 times the volume of solution. Warm the solution in a 40 &#176;C water bath or in a microwave for 10&#8722;20 s to melt the solution. Pipette the melted agarose around the edges of the gel. After allowing the agarose to harden at RT or 4 &#176;C, add water to the sample to prevent dehydration.</li>
</ol>

<ol>
	<li>Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Far-Field+Optical+Nanoscopy.">Far-Field Optical Nanoscopy.</a> Science. 316 (5828), 1153-1158 (2007).</li><li>Combs, C. A., Shroff, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy:+A+concise+guide+to+current+imaging+methods.">Fluorescence microscopy: A concise guide to current imaging methods.</a> Current Protocols in Neuroscience. 79 (1), 2.1.1-2.1.25 (2017).</li><li>Schermelleh, L., Heintzmann, R., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+super-resolution+fluorescence+microscopy.">A guide to super-resolution fluorescence microscopy.</a> Journal of Cell Biology. 190 (2), 165-175 (2010).</li><li>Chen, F., Tillberg, P. W., Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+microscopy.">Expansion microscopy.</a> Science. 347 (6221), 543-548 (2015).</li><li>Tillberg, P. W., 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=Protein-retention+expansion+microscopy+of+cells+and+tissues+labeled+using+standard+fluorescent+proteins+and+antibodies.">Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies.</a> Nature Biotechnology. 34, 987-992 (2016).</li><li>Chozinski, T. 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=Expansion+microscopy+with+conventional+antibodies+and+fluorescent+proteins.">Expansion microscopy with conventional antibodies and fluorescent proteins.</a> Nature Methods. 13 (6), 485-488 (2016).</li><li>Ku, T., 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=Multiplexed+and+scalable+super-resolution+imaging+of+three-dimensional+protein+localization+in+size-adjustable+tissues.">Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues.</a> Nature Biotechnology. 34 (9), 973-981 (2016).</li><li>Truckenbrodt, S., Maidorn, M., Crzan, D., Wildhagen, H., Kabatas, S., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X10+expansion+microscopy+enables+25-nm+resolution+on+conventional+microscopes.">X10 expansion microscopy enables 25-nm resolution on conventional microscopes.</a> EMBO Reports. 19 (9), e45836 (2018).</li><li>Asano, S. M., 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=Expansion+Microscopy:+Protocols+for+Imaging+Proteins+and+RNA+in+Cells+and+Tissues.">Expansion Microscopy: Protocols for Imaging Proteins and RNA in Cells and Tissues.</a> Current Protocols in Cell Biology. 80 (1), 1-41 (2018).</li><li>Chen, F., 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=Nanoscale+imaging+of+RNA+with+expansion+microscopy.">Nanoscale imaging of RNA with expansion microscopy.</a> Nature Methods. 13 (8), 679-684 (2016).</li><li>Tsanov, 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=SmiFISH+and+FISH-quant+-+A+flexible+single+RNA+detection+approach+with+super-resolution+capability.">SmiFISH and FISH-quant - A flexible single RNA detection approach with super-resolution capability.</a> Nucleic Acids Research. 44 (22), e165 (2016).</li><li>Wang, G., Moffitt, J. R., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+imaging+of+high-density+libraries+of+RNAs+with+MERFISH+and+expansion+microscopy.">Multiplexed imaging of high-density libraries of RNAs with MERFISH and expansion microscopy.</a> Scientific Reports. 8 (1), 1-13 (2018).</li><li>Chang, J. 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=Iterative+expansion+microscopy.">Iterative expansion microscopy.</a> Nature Methods. 14 (6), 593-599 (2017).</li><li>Cipriano, B. 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=Superabsorbent+hydrogels+that+are+robust+and+highly+stretchable.">Superabsorbent hydrogels that are robust and highly stretchable.</a> Macromolecules. 47 (13), 4445-4452 (2014).</li><li>Truckenbrodt, S., Sommer, C., Rizzoli, S. O., Danzl, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+optimization+in+X10+expansion+microscopy.">A practical guide to optimization in X10 expansion microscopy.</a> Nature Protocols. 14 (3), 832-863 (2019).</li><li>Zhao, Y., 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=Nanoscale+imaging+of+clinical+specimens+using+pathology-optimized+expansion+microscopy.">Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy.</a> Nature Biotechnology. 35 (8), 757-764 (2017).</li></ol>

YZ and OB are two of the inventors who have filed for and obtained patent protection on a subset of the technologies described here (US patents US20190064037A1, WO2018157074A1, and WO2018157048A1).

Here, we present the ExPath protocol16, a variant of proExM5 that can be applied to the most common types of clinical biopsy samples used in pathology, including FFPE, H&#38;E stained, and fresh-frozen specimens on glass slides. Format conversion, antigen retrieval, and immunostaining of the specimens follow commonly used protocols that are not specific to ExPath. Unlike the original proExM protocol9, ExPath relies on a higher concentration of EDTA i...

Investigating the molecular organization of tissues in a three-dimensional (3D) context can provide new understanding of biological functions and disease development. However, these nanoscale environments are beyond the resolution capabilities of conventional diffraction limited microscopes (200&#8722;300 nm), where the minimal resolvable distance, d is defined by d &#945; &#955;/NA. Here &#955; is the wavelength of light and NA is the numerical aperture (NA) of the imaging system. Recently, direct visualization of fluorescently labeled molecules has been made possible by newly developed super-resolution imaging techniques1,2,3, including stimulated emission depletion (STED), photo-activated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and structured illumination microscopy (SIM). Although these imaging techniques have revolutionized understanding of biological function at the nanoscale, in practice, they often rely on expensive and/or specialized equipment and image processing steps, can have slower acquisition time comparing to conventional optical imaging, require fluorophores with specific characteristics (such as photo-switching capability and/or high photostability). In addition, it remains a challenge to perform 3D super-resolution imaging on tissue specimens.
Expansion microscopy (ExM), first introduced in 20154, provides an alternative means of imaging nanoscale features (&#60;70 nm) by physically expanding preserved samples embedded in a swellable polyelectrolyte hydrogel. Here, key biomolecules and/or labels are anchored in situ to a polymer network that can be isotopically expanded after chemical processing. Because the physical expansion increases the total effective resolution, molecules of interest can then be resolved using conventional diffraction-limited imaging systems. Since the publication of the original protocol, where custom synthesized fluorescent labels were anchored to the polymer network4, new strategies have been used to directly anchor proteins (protein retention ExM, or proExM)5,6,7,8,9 and RNA9,10,11,12 to the hydrogel, and increase physical magnification through iterative expansion13 or adapting gel chemistry8,14,15.
Here we present an adapted version of proExM, called expansion pathology (ExPath)16, which has been optimized for clinical pathology formats. The protocol converts clinical samples, including formalin-fixed paraffin-embedded (FFPE), hematoxylin and eosin (H&#38;E) stained, and fresh-frozen human tissue specimens mounted on glass slides, into a state compatible with ExM. Proteins are then anchored to the hydrogel and mechanical homogenization is performed (Figure 1)16. With a 4-fold linear expansion of the samples, multicolor super-resolution (~70 nm) images can be obtained using a conventional confocal microscope having only a ~300 nm resolution and can also be combined with other super-resolution imaging techniques.

<table>
	<tbody>
		<tr>
			<td>4-hydroxy-TEMPO (4HT)</td>
			<td>Sigma Aldrich</td>
			<td>176141</td>
			<td>Inhibitor</td>
		</tr>
		<tr>
			<td>6-well glass-bottom plate (#1.5 coverglass)</td>
			<td>Cellvis</td>
			<td>P06-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fischer Scientifc</td>
			<td>A18-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Sigma Aldrich</td>
			<td>A8887</td>
			<td></td>
		</tr>
		<tr>
			<td>Acryloyl-X, SE (AcX)</td>
			<td>Invitrogen</td>
			<td>A20770</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fischer Scientifc</td>
			<td>BP160-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma Aldrich</td>
			<td>A3678</td>
			<td>Initiatior</td>
		</tr>
		<tr>
			<td>Anti-ACTN4 antibody produced in rabbit</td>
			<td>Sigma Aldrich</td>
			<td>HPA001873</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Collagen IV antibody produced in mouse</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-59814</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Vimentin antibody produced in chicken</td>
			<td>Abcam</td>
			<td>ab24525</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua Hold II hydrophobic pen</td>
			<td>Scientific Device</td>
			<td>980402</td>
			<td></td>
		</tr>
		<tr>
			<td>Breast Common Disease Tissue Array</td>
			<td>Abcam</td>
			<td>ab178113</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (1 mg/mL)</td>
			<td>Thermo Scientific</td>
			<td>62248</td>
			<td>Nuclear stain</td>
		</tr>
		<tr>
			<td>Diamond knife No. 88 CM</td>
			<td>General Tools</td>
			<td>31116</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic 
			acid (EDTA) 0.5 M</td>
			<td>VWR</td>
			<td>BDH7830-1</td>
			<td></td>
		</tr>
		<tr>
			<td>FFPE Kidney Sample</td>
			<td>USBiomax</td>
			<td>HuFPT072</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Chicken IgY (H+L), Highly Cross-Adsorbed CF488A</td>
			<td>Biotium</td>
			<td>20020</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Chicken IgY (H+L), Highly Cross-Adsorbed CF633</td>
			<td>Biotium</td>
			<td>20121</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Alexa Fluor 546</td>
			<td>Invitrogen</td>
			<td>A11010</td>
			<td></td>
		</tr>
		<tr>
			<td>MAXbind Staining Medium</td>
			<td>Active Motif</td>
			<td>15253</td>
			<td>Can be substituted with non-commercial staning buffer of choice.</td>
		</tr>
		<tr>
			<td>MAXblock Blocking Medium</td>
			<td>Active Motif</td>
			<td>15252</td>
			<td>Can be substituted with non-commercial blocking buffer of choice.</td>
		</tr>
		<tr>
			<td>MAXwash Washing Medium</td>
			<td>Active Motif</td>
			<td>15254</td>
			<td>Can be substituted with non-commercial washing buffer of choice.</td>
		</tr>
		<tr>
			<td>Micro cover Glass #1 (24x60mm)</td>
			<td>VWR</td>
			<td>48393 106</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro cover Glass #1.5 (24x60mm)</td>
			<td>VWR</td>
			<td>48393 251</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;- 
			Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma Aldrich</td>
			<td>T9281</td>
			<td>Accelerator</td>
		</tr>
		<tr>
			<td>N,N&#8242;-Methylenebisacrylamide</td>
			<td>Sigma Aldrich</td>
			<td>M7279</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson Immunoresearch</td>
			<td>005-000-121</td>
			<td>For preparing blocking buffer. Dependent on animal host of secondary antibodies.</td>
		</tr>
		<tr>
			<td>Nunclon 4-Well x 5 mL MultiDish Cell Culture Dish</td>
			<td>Thermo Fisher</td>
			<td>167063</td>
			<td>Multi-well plastic culture dish</td>
		</tr>
		<tr>
			<td>Nunclon 6-Well Cell Culture Dish</td>
			<td>Thermo Fisher</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc 15mL&#160;Conical</td>
			<td>Thermo Fisher</td>
			<td>339651</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc 50mL&#160;Conical</td>
			<td>Thermo Fisher</td>
			<td>339653</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paint brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH Meter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS), 10x Solution</td>
			<td>Fischer Scientifc</td>
			<td>BP399-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Petri Dish (100 mm)</td>
			<td>Fischer Scientifc</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K (Molecular Biology Grade)</td>
			<td>Thermo Scientific</td>
			<td>EO0491</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Fischer Scientifc</td>
			<td>12640</td>
			<td></td>
		</tr>
		<tr>
			<td>Safelock Microcentrifuge Tubes 1.5 mL</td>
			<td>Thermo Fisher</td>
			<td>3457</td>
			<td></td>
		</tr>
		<tr>
			<td>Safelock Microcentrifuge Tubes 2.0 mL</td>
			<td>Thermo Fisher</td>
			<td>3459</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acrylate</td>
			<td>Sigma Aldrich</td>
			<td>408220</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>S6191</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate tribasic dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C8532-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fischer Scientifc</td>
			<td>BP152-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat germ agglutinin labeled with CF640R</td>
			<td>Biotium</td>
			<td>29026</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>Sigma Aldrich</td>
			<td>214736</td>
			<td></td>
		</tr>
	</tbody>
</table>

nanoscopic imaging of human tissue sections via physical and isotropic expansion

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, the fundamental physical diffraction limit prevents interrogation of nanoscale anatomy and subtle pathological changes when using conventional optical imaging approaches. Here, we describe a simple and inexpensive protocol, called expansion pathology (ExPath), for nanoscale optical imaging of common types of clinical primary tissue specimens, including both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. This method circumvents the optical diffraction limit by chemically transforming the tissue samples into tissue-hydrogel hybrid and physically expanding them isotropically across multiple scales in pure water. Due to expansion, previously unresolvable molecules are separated and thus can be observed using a conventional optical microscope.

If the protocol has been successfully carried out (Figure 1), samples will appear as a flat and transparent gel after mechanical homogenization (Figure 3A) and can expand by a factor of 3&#8722;4.5x in water (Figure 3B), providing an effective resolution of ~70 nm depending on the final expansion factor and imaging system used5,16. <strong c...

Watch this Scientific Journal Video about Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion at JoVE.com

Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion

Nanoscale imaging of clinical tissue samples can improve understanding of disease pathogenesis. Expansion pathology (ExPath) is a version of expansion microscopy (ExM), modified for compatibility with standard clinical tissue samples, to explore the nanoscale configuration of biomolecules using conventional diffraction limited microscopes.

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, ...

nanoscopic-imaging-human-tissue-sections-via-physical-isotropic

Research

JoVE Journal

Medicine

7.5K Views.  Carnegie Mellon University. Nanoscale imaging of clinical tissue samples can improve understanding of disease pathogenesis. Expansion pathology (ExPath) is a version of expansion microscopy (ExM), modified for compatibility with standard clinical tissue samples, to explore the nanoscale configuration of biomolecules using conventional diffraction limited microscopes.

Video: Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. Therefore, the ability of the blood vessels to dilate or constrict, known as Cerebral-Vascular-Reactivity (CVR), represents an important domain of vascular function. An imaging marker representing this dynamic property will provide new information of cerebral vessels under normal and diseased conditions such as stroke, dementia, atherosclerosis, small vessel diseases, brain tumor, traumatic brain injury, and multiple sclerosis. In order to perform this type of measurement in humans, it is necessary to deliver a vasoactive stimulus such as CO2 and/or O2 gas mixture while quantitative brain magnetic resonance images (MRI) are being collected. In this work, we presented a MR compatible gas-delivery system and the associated protocol that allow the delivery of special gas mixtures (e.g., O2, CO2, N2, and their combinations) while the subject is lying inside the MRI scanner. This system is relatively simple, economical, and easy to use, and the experimental protocol allows accurate mapping of CVR in both healthy volunteers and patients with neurological disorders. This approach has the potential to be used in broad clinical applications and in better understanding of brain vascular pathophysiology. In the video, we demonstrate how to set up the system inside an MRI suite and how to perform a complete experiment on a human participant.

Non-invasive imaging of the brain vasculature&#8217;s ability to dilate or constrict may allow a better understanding of cerebrovascular pathophysiology in various neurological diseases. The present report describes a reproducible and patient-comfortable protocol to perform vascular reactivity imaging in humans using magnetic resonance imaging (MRI).

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. ...

High-definition Fourier Transform Infrared (FT-IR) Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

High-definition Fourier Transform Infrared (FT-IR) spectroscopic imaging is an emerging approach to obtain detailed images that have associated biochemical information. FT-IR imaging of tissue is based on the principle that different regions of the mid-infrared are absorbed by different chemical bonds (e.g., C=O, C-H, N-H) within cells or tissue that can then be related to the presence and composition of biomolecules (e.g., lipids, DNA, glycogen, protein, collagen). In an FT-IR image, every pixel within the image comprises an entire Infrared (IR) spectrum that can give information on the biochemical status of the cells that can then be exploited for cell-type or disease-type classification. In this paper, we show: how to obtain IR images from human tissues using an FT-IR system, how to modify existing instrumentation to allow for high-definition imaging capabilities, and how to visualize FT-IR images. We then present some applications of FT-IR for pathology using the liver and kidney as examples. FT-IR imaging holds exciting applications in providing a novel route to obtain biochemical information from cells and tissue in an entirely label-free non-perturbing route towards giving new insight into biomolecular changes as part of disease processes. Additionally, this biochemical information can potentially allow for objective and automated analysis of certain aspects of disease diagnosis.

High-definition Fourier Transform Infrared FT-IR Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

Fourier Transform Infrared (FT-IR) spectroscopic imaging is a fast and label-free approach to obtain biochemical data sets of cells and tissues. Here, we demonstrate how to obtain high-definition FT-IR images of tissue sections towards improving disease diagnosis.

High-definition Fourier Transform Infrared (FT-IR) spectroscopic imaging is an emerging approach to obtain detailed images that have associated ...

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of the complexities of biological indices in white adipose tissue. Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding. Nevertheless, side effects, such as flash fires and skin lesions in the tissue, have been reported after diathermy. Therefore, we aimed to standardize a surgical abdominal adipose tissue biopsy performed under local anesthesia using a non-diathermy method. We conducted 115 subcutaneous adipose tissue biopsies in healthy men using a non-diathermy abdominal surgical biopsy method. Our results showed three cases of excessive post-operation bleeding out of 115 operations (2.61%).In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men at the bedside, with minimal side effects.

We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

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

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

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