Name: spot the correct tissue every time in multi-tissue blocks
Uploaded: 2015-05-31T14:00:00.000+00:00
Duration: 6 min 53 s
Description: Watch this Scientific Journal Video about SpOT the Correct Tissue Every Time in Multi-tissue Blocks at JoVE.com

The authors would like to thank Dr. Brent Harris for providing critical review of the manuscript. These studies were conducted at the Lombardi Comprehensive Cancer Center Histopathology &#38; Tissue Shared resource which is supported in part by NIH/NCI grant P30-CA051008. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.

1. Construction of the SpOT
<ol>
	<li>Add 50 mg of Bovine Serum Albumin (BSA) to 1 ml tissue marking dye in a 15 ml conical tube or 2 ml microfuge tube and vortex for 1 min or until completely dissolved. 
	Note: For this protocol, use biochemical Grade BSA. While other grades and purities have not been tested, it is expected that grade and purity would not have a significant impact on the success of the SpOT.</li>
	<li>Heat 9 ml hydroxyethyl agarose processing gel in a 15 ml conical tube in a microwave at 30% power for 10 sec increments until the gel is completely melted.</li>
	<li>Combine the melted processing gel and dye-BSA solution in a single 15 ml conical tube and mix thoroughly with a pipette. Vortex the solution.</li>
	<li>Place the conical tube in a refrigerator for a few hrs or until solid.</li>
	<li>Use a long, thin, metal spatula or probe to gently release the colored gel plug from the conical tube.</li>
	<li>Use a scalpel or razor blade to cut the gel plug transversely into 5 mm thick sections. Remove the rounded ends and dispose as waste (Figure 2A).</li>
	<li>Place the gel plug sections flat in histology cassettes and hold in 70% ethanol for 1 hr. Change the 70% ethanol solution every 2-4 hr for at least 3 changes over a 24 hr period.</li>
	<li>Manually process the gel sections through an ethanol series (1 hr each: 70% ethanol, 80% ethanol, 95% ethanol, 100% ethanol (3x changes), then clear in clearing solution 3x for 1 hr each (e.g., aliphatic hydrocarbons (xylenes substitute)) were used in this protocol, xylenes are also acceptable), and infiltrate with molten paraffin (3x for 1 hr each), either manually or in a tissue processer. 
	Note: Prior to full dehydration in 100% ethanol, the dye may leak from the gel plug which could affect other tissues and the liquid reagents in an automated tissue processor. As such, manual rehydration is highly recommended, however automated processing is equally effective.</li>
	<li>Embed multiple processed gel sections using standard paraffin embedding methodology (Figure 2B). Allow blocks to cool and harden and then come to room temperature.</li>
	<li>Use a dermal punch needle of desired size to remove SpOT cores from the embedded gel sections until the block is exhausted (Figure 2C). A single block may provide up to 20 x 2 mm2 cores. Store the cores in a cool area. Use dermal punch in this protocol see Figures, a 1.5 mm (Figures 1D, and 2D) or a 2 mm (Figures 1B, 1C, and 2C).</li>
</ol>
2. Using SpOTs to Orient Non-arrayed Blocks
<ol>
	<li>Create a tissue orientation diagram to indicate the desired locations and identities of all of the individual tissue pieces to be embedded together, and the location of the SpOT. 
	NOTE: The tissue orientation diagram is an illustrated map that includes the physical locations of all tissue pieces labeled with identifiers and the location of the SpOT. It can be created electronically or can be hand-drawn. The diagram should be created using whatever method best suits the technician and researcher using the final product. This map will serve as the guide for the researcher so samples can be easily identified during microscopic analysis. The map used in this protocol was hand-drawn. It depicts a tissue block and glass microscope slide with each piece of tissue and the SpOT drawn and labeled in the same position as the actual tissue block and slide.</li>
	<li>Process the tissue pieces normally, ensuring that they remain properly identified throughout the grossing and processing steps. For this protocol, process the tissues for 1 hr each in: 70% ethanol, 80% ethanol, 95% ethanol, 100% ethanol (x3 changes), aliphatic hydrocarbons (xylenes substitute) (x3 changes), and molten tissue infiltration paraffin at 60 &#176;C (x3 changes). 
	NOTE: All tissues should be processed following standard histology laboratory procedures. This may be variable due to individual laboratory procedure, tissue size and type, but this will not affect the ability to use the SpOT as an orientation tool.</li>
	<li>Arrange tissue pieces on the embedding station in the correct assigned locations according to the tissue orientation diagram. Use the tissue orientation diagram as a reference to guide the technician on how to arrange the SpOT and tissue pieces in every block. 
	NOTE: In this protocol the embedding technician put the hand-drawn orientation map next to the embedding station and arranged the tissue pieces on the embedding station in exactly the same arrangement as drawn on the diagram. This ensures that all tissue pieces remain correctly identifiable in the final product. This is absolutely critical to the successful use of multi-tissue blocks.</li>
	<li>Ensure the SpOT core is taller than all tissue pieces to be embedded in the block.</li>
	<li>Embed the tissue pieces and then the SpOT on end (transverse) in the proper locations according to the tissue orientation diagram using standard embedding methodology. As needed, hold the SpOT(s) upright with forceps until the paraffin has solidified enough that the SpOT(s) will not fall over (1-2 min).</li>
	<li>Section and stain paraffin sections with the SpOT using standard methodology.
	<ol>
		<li>Allow the sections to float on a water bath at 40 &#176;C and adhered to a positively charged glass slide (uncharged slides have not been tested). Dry the slides in a drying oven at 60 &#176;C for at least 20 min.</li>
		<li>Place the slides in an automatic stainer and processed through the following reagents: xylenes (xylenes 1-5 min, xylenes 2 and 3-2 min, each), 100% ethanol (2 min), 95% ethanol (1 min), 80% ethanol (30 sec), 70% ethanol (30 sec), running tap water (2 min), hematoxylin (1.5 min), running tap water (3 min), acid alcohol (1 min), running tap water (2 min), bluing reagent (1 min), running water (2 min), 80% ethanol (30 sec), eosin (1 min), running water (10 sec), 80% ethanol (1 min), 100% ethanol (3 changes, 2 min each), xylenes (3 changes, 30 sec each).</li>
		<li>Cover the slides with glass coverslips, mounted with mounting medium. 
		NOTE: In this protocol, the histotechnician sectioned the paraffin blocks on a microtome in 5 &#181;m sections. For this protocol we used an automatic stainer, however equal results can be obtained via manual staining.</li>
	</ol>
	</li>
</ol>
3. SpOT in TMAs (Manual TMA Kit)
<ol>
	<li>Create a tissue orientation diagram, as described in 2.1 and assign the desired location(s) of the SpOT.</li>
	<li>Fill the TMA mold with melted paraffin at an embedding station. As the mold is cooling, embed the SpOT(s) on end in the margins of the TMA mold at the desired locations indicated by the tissue orientation diagram. As needed, hold the SpOT(s) upright with forceps until the paraffin has solidified enough that the SpOT(s) will not fall over (1-2 min).</li>
	<li>Assemble the TMA cores according to manufacturer&#8217;s directions and with regard to the location of the SpOT(s) in the array margins (Figure 2D), and follow standard sectioning and staining protocols. 
	NOTE: Please see step 2.6 to see the sectioning and staining methods used in this protocol.</li>
</ol>

<ol>
	<li>Jawhar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+microarray:+a+rapidly+evolving+diagnostic+and+research+tool.">Tissue microarray: a rapidly evolving diagnostic and research tool.</a> Ann. Saudi. Med. 29 (2), 123-127 (2009).</li><li>Dhir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+microarrays:+an+overview.">Tissue microarrays: an overview.</a> Methods Mol. Bio. 441, 91-103 (2008).</li><li>Parsons, M., Grabsch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+tissue+microarrays.">How to make tissue microarrays.</a> Diagn. Histopath. 15 (3), 142-150 (2009).</li><li>Saxena, R., Bade, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+7+Tissue+Microarray+–+Construction+and+Quality+Assurance.">Ch. 7 Tissue Microarray – Construction and Quality Assurance.</a> Immunohistochemical Staining Methods 6th ed. , (2013).</li><li>Nocito, A., Kononen, J., Kallioniemi, O., Sauter, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+microarrays+(TMAs)+for+high-throughput+molecular+pathology+research.">Tissue microarrays (TMAs) for high-throughput molecular pathology research.</a> Int. J. Cancer. 94, 1-5 (2001).</li><li>Rangel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tissue+microarray:+helpful+hints.">The tissue microarray: helpful hints.</a> J.Histotech. 25 (2), 93-100 (2002).</li><li>Hammer, A., Williams, B., Dietz, H., Hamilton-Dutoit, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+immunophenotyping+of+43+ferret+lymphomas+using+tissue+microarray+technology.">High-throughput immunophenotyping of 43 ferret lymphomas using tissue microarray technology.</a> Vet. Path. 44, 196-203 (2007).</li><li>Eckel-Passow, J., Lohse, C., Sheinin, Y., Crispen, P., Krco, C., Kwon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+microarrays:+one+size+does+not+fit+all.">Tissue microarrays: one size does not fit all.</a> Diagn Pathol. 5, 48-57 (2010).</li><li>Lin, Y., Hatem, J., Wang, J., Quinn, A., Hicks, D., Tang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+microarray-based+immunohistochemical+study+can+significantly+underestimate+the+expression+of+HER2+and+progesterone+receptor+in+ductal+carcinoma+in+situ+of+the+breast.">Tissue microarray-based immunohistochemical study can significantly underestimate the expression of HER2 and progesterone receptor in ductal carcinoma in situ of the breast.</a> Biotech. Histochem. 86 (5), 345-350 (2011).</li><li>Wampfler, 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=Determining+the+optimal+numbers+of+cores+based+on+tissue+microarray+antibody+assessment+in+non-small+cell+lung+cancer.">Determining the optimal numbers of cores based on tissue microarray antibody assessment in non-small cell lung cancer.</a> J Cancer Sci. Ther. 3, 120-124 (2011).</li><li>Quagliata, L., Schlageter, M., Quintavalle, C., Tornillo, L., Terracciano, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+new+players+in+hepatocarcinogenesis:+Limits+and+opportunities+of+using+tissue+microarray+(TMA).">Identification of new players in hepatocarcinogenesis: Limits and opportunities of using tissue microarray (TMA).</a> Microarray. 3, 91-102 (2014).</li><li>Miettinen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+generating+multitissue+blocks+without+special+equipment.">A simple method for generating multitissue blocks without special equipment.</a> Immunohistochem. Mol. Morphol. 20 (4), 410-412 (2012).</li><li>Jones, M., Calabresdi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agar-gelatin+for+embedding+tissues+prior+to+paraffin+processing.">Agar-gelatin for embedding tissues prior to paraffin processing.</a> BioTechniques. 42, 569-570 (2007).</li><li>Carson, F., Hladik, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histotechnology: A Self-Instructional Text. , (2009).</li><li>Suvarna, S., Layton, C., Bancroft, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bancroft&#8217;s Theory and Practice of Histological Techniques. 7th ed. , (2013).</li></ol>

The authors have nothing to disclose.

Successful preparation and utilization of the SpOTs requires careful adherence to a few technical steps. Melting of the hydroxyethyl agarose should be done slowly and at low heat. Melting quickly at high heat can result in some breakdown of the agarose for less than optimal results. When cutting the dye-laden plugs into pieces for processing, ensure that the thickness of each piece is greater than 4.5 mm and does not exceed 7 mm. The rounded ends are removed as waste as they are not uniformly 5 mm in thickness. Pieces less than 5 mm will result in the creation of shorter SpOTs that risk being exhausted by sectioning prior to the exhaustion of adjacent tissue material. To ensure the SpOT is present through the entirety of the block, the pieces need to be at least 4.5-5 mm. To ensure proper infiltration of wax, however, the pieces should not exceed 7 mm. When dehydrating the dye pieces for wax infiltration, the dye will leach slightly out of the pieces in the lower concentrations of ethanol. The leaching does not impact the final product, the SpOTs will still be intensely dyed and clearly visible. The leaching could impact other tissues, though, so tissue sections should not be processed in the same baths as the dye pieces until the pieces have reached 100% ethanol.
When placing the SpOTs into the arrayed blocks, ensure that all other tissue placement is complete prior to adding the SpOT. Since the SpOT is infiltrated with paraffin wax, the molten wax of the recipient block can begin to melt the SpOT core if it is allowed to sit for too long. Arrange all tissue pieces first, add the SpOT core and immediately transfer the block to a cold plate to prevent any melting of the SpOT.
The SpOT can be modified to fit into most histology lab workflows. The color of the dye can be changed for optimal contrast in each application. Red or black dye produces clear high contrast on IHC stained slides, while the red SpOT is not as eye catching in an H&#38;E stained slide of arrayed kidney sections. For H&#38;E slides, green or yellow SpOTs tend to provide higher contrast. Further, under high heat during processes such as heat induced antigen retrieval for immunohistochemistry, the hydroxyethyl agarose melts away. BSA is added to the dye during SpOT preparation to maintain a brightly colored SpOT on the slide even after high heat retrieval methods. If the slides will not be exposed to high heat, the BSA can be omitted for faster SpOT production.
The most critical aspects of the use of SpOT are in the creation and maintenance of a clear and concise orientation map and the faithful adherence to the map when placing the SpOT. When used properly, the SpOT reduces confusion over tissue arrangement and simplifies communication between technicians and researchers, as the embedding technician can simply indicate the location of the SpOT and the relation of the tissues to it on a map. SpOT is clearly visible in all steps of slide preparation allowing for highly consistent section mounting on slides for easier downstream analysis. In contrast to physiological markers, the SpOT is clearly visible in every section and does not modify the tissue in any way, preventing the introduction of artifacts or obstruction of morphology. Unlike the method of embedding the tissues asymmetrically, the SpOT allows technicians and researchers flexibility in the arrangement of tissues to optimize visual analysis under the microscope. In TMAs, the SpOT can be placed outside the main array, eliminating the need for blank spaces, asymmetric assemblies, or beacon cores, allowing the entire array to be composed of valuable tissue sections. The SpOT can also be used to indicate anatomical direction (e.g., distal or proximal) of tissue samples. The SpOT is a simple and low cost solution for easy and consistent orientation of tissue samples during construction and analysis of multi-tissue blocks and TMAs.

The ability to embed tissue samples from multiple individuals in a single paraffin block enables easy side-by-side comparison between treatments and individuals, eliminates variability between slides, and reduces the cost and workload of sectioning and staining specimens. These multi-tissue blocks are typically produced as either tissue microarrays (TMA) or paraffin blocks containing tissue from multiple individuals in a non-array layout. Maintenance of sample identity is critical to the success of any multi-tissue analysis. Researchers have been using TMAs since their development to improve efficiency of analysis, reduce variation between slides, conserve valuable tissue resources, and reduce the time and cost of experiments1. Correct orientation of TMAs can be accomplished using a variety of methods, including blank spaces, rows or columns between tissue cores2-4, asymmetric arrangement of core groups3, 5 (e.g., control vs. treatment), &#8220;beacon&#8221; cores6, and designated orientation cores located outside the TMA matrix3. Although these methods work well for TMAs, most consume valuable TMA core space and TMAs with gaps and spaces as identifiers may become confusing as tissue cores are exhausted over time. Additionally, these methods are not appropriate for use in multi-tissue blocks without an array format because they rely on anomalies in the tightly ordered pattern of uniform microarray cores as identifiers. Most non-arrayed multi-tissue blocks must accommodate non-uniform tissues and, by definition, do not display the structured array grid that makes these aberrations stand out as landmarks.
Although there are many advantages to using a TMA, one of the largest disadvantages is the small size of the cores which is not always representative of largely heterogeneous tissue1. Non-arrayed multi-tissue blocks provide many of the benefits of a TMA, but contain larger tissue samples, or entire organs from animal studies. Tissue microarrays using single cores have varying concordance with whole tissue sections based on the protein of interest and many require multiple cores for increased concordance7-11. Due to the complexity and heterogeneous nature of some biomarker phenotypes, TMAs using even large numbers of cores (&#62;10) can still be insufficient and may require methods other than microarrays for analysis8. Additionally, TMA construction is time consuming, technically demanding, and requires the initial cost and investment in or access to a tissue arrayer. Non-arrayed multi-tissue blocks can be made in any basic laboratory with significantly less time and effort and is a valid alternative for studies that require more tissue than arrays allow or as a means to cut costs and simplify analysis.
Non-arrayed multi-tissue blocks similarly require a reliable orientation marker to track sample identification, however, the development of these markers has been limited. Much of the literature describing tissue orientation focuses on correct physiological orientation for embedding individual tissue pieces, such as tattooing the tissue with ink12, pre-embedding tissues in agar-gelatin prior to processing13, and marking certain tissues with notches14 or sutures15. Although functional, these methods are not ideal as markers in multi-tissue blocks due to their limitations. A suture will be sectioned through quickly and may not be visible in every section. Pre-embedding techniques using agar-gelatin may keep the tissue in proper orientation during processing and embedding, but does not provide a visual cue to differentiate between multiple samples in the paraffin block and slides. Notches or dye on the tissue may complicate analysis or occlude important morphological details. Alternatively, tissue identity in a non-arrayed multi-tissue block can be maintained through embedding of tissue pieces in an asymmetric arrangement, but this requires 3 or more tissue pieces and may not allow for optimal arrangement of tissues for analysis.
The Specimen Orientation Tag (SpOT) has been developed as an easy and inexpensive method to clearly identify tissues in multi-tissue blocks and offers many benefits over existing orientation methods. The SpOT is a small, colorful, core composed of Hydroxyethyl agarose processing gel and tissue marking dye, and infiltrated with paraffin (Figure 2C). The SpOT core is embedded on end next to a single tissue in a multi-tissue paraffin block and appears as a brightly colored dot in the block and in every section (Figure 1A-D), clearly indicating the correct orientation of the block and sections for easy tissue identification.

<table><tbody><tr><td>Histogel Specimen Processing Gel</td><td>Thermo Scientific</td><td>HG-4000-012</td><td>&lt;a href=&quot;http://www.thermoscientific.com/en/product/richard-allan-scientific-histogel-specimen-processing-gel.html&quot; target=&quot;_blank&quot;&gt;http://www.thermoscientific.com/en/product/richard-allan-scientific-histogel-specimen-processing-gel.html&lt;/a&gt;</td></tr><tr><td>Tissue Marking Dye</td><td>Triangle Biomedical Sciences, Inc.</td><td>TMD-5</td><td>Any tissue marking dye would most likely be sufficient.</td></tr><tr><td>Arraymold Kit A 2 mm (60 core)</td><td>Arraymold</td><td>20015A</td><td>Any manual tissue arrayer would work similarly.</td></tr></tbody></table>

spot the correct tissue every time in multi-tissue blocks

Multi-tissue paraffin blocks provide high throughput analysis with increased efficiency, experimental uniformity, and reduced time and cost. Tissue microarrays make up the majority of multi-tissue paraffin blocks, but increasingly, researchers are using non-arrayed blocks containing larger tissues from multiple individuals which can provide many of the advantages of tissue microarrays without substantial investment in planning and equipment. A critical component of any multi-tissue analysis is the orientation method used to identify each individual tissue. Although methods exist to maintain proper orientation and identification of tissues in multi-tissue blocks, most are not well-suited to non-arrayed blocks, may consume valuable space within an array and/or are difficult to produce in the standard histology laboratory. The Specimen Orientation Tag (SpOT) is a simple, low cost orientation tool that is clearly visible in paraffin blocks and all tissue sections for reliable specimen identification in arrayed and non-arrayed layouts. The SpOT provides advantages over existing orientation methods for non-arrayed blocks as it does not require any direct modification to the tissue and allows for flexibility in the arrangement of tissue pieces.

The SpOT appears as a round, brightly colored dot in the paraffin block (Figure 1A and 2D), in all paraffin sections, and remains on the glass slide through the H&#38;E or IHC staining procedure (Figures 1B-1D, and 2D). This obvious visual cue aids both the histotechnician and researcher in identifying each individual tissue piece and simplifies communication as the histotechnician can use this visual cue to indicate the arrangement of the tissue pieces to the researcher collecting data from the slide at the microscope. The tissue orientation diagram should be included with the slides provided to the researcher and explained in detail so there will be no confusion during microscopic analysis. Figure 2A shows the results of sectioning the solidified, colored agarose gel plugs. Figure 2B shows the result of embedding the sectioned agarose gel plugs in a paraffin block. Figure 2C shows the results of using a dermal punch to produce the SpOT cores.
<img alt="Figure 1" src="/files/ftp_upload/52868/52868fig1highres.jpg" width="700" /> 
Figure 1: SpOT cores are easily created and readily visible in tissue blocks and slides. (A) SpOTS are clearly visible in the embedded block. (B) SpOTS allow for easy orientation and tissue identification for multi-tissue blocks containing similar appearing tissues from different individuals/treatment groups. (C) SpOTs allow every core of a Tissue Microarray to be utilized for valuable tissue samples. (D) Microscope view of SpOT adjacent to a TMA core. Arrows indicate the SpOT. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52868/52868fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52868/52868fig2highres.jpg" width="700" /> 
Figure 2: Preparation of SpOT cores. (A) Solidified Hydroxyethyl agarose plugs are released from Eppendorf tubes and cut transversely into 5 mm slices. (B) Multiple slices are embedded in a single paraffin block and (C) a dermal punch is used to remove SpOTs cores. (D) SpOTs are embedded in the margins of a Tissue Microarray. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52868/52868fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about SpOT the Correct Tissue Every Time in Multi-tissue Blocks at JoVE.com

SpOT the Correct Tissue Every Time in Multi-tissue Blocks

The purpose of the Specimen Orientation Tag (SpOT) is to function as an orientation tool to aid in individual tissue identification in multi-tissue paraffin blocks. These protocols demonstrate how it is constructed easily from common, low-cost histology materials and serves as a reliable visual marker in paraffin blocks and sections.

Multi-tissue paraffin blocks provide high throughput analysis with increased efficiency, experimental uniformity, and reduced time and cost. Tissue ...

spot-the-correct-tissue-every-time-in-multi-tissue-blocks

Nanyang Technological University

Research

JoVE Journal

Biology

11.7K Views.  Frederick National Laboratory for Cancer Research.  Het doel van het Specimen Orientation Tag (spot) is gefunctioneerd als leidraad ter te helpen bij individuele identificatie weefsel in meerdere weefsels paraffineblokken. Deze protocollen laten zien hoe dit is opgebouwd uit zachte, low-cost histologie materiaal en dient als een betrouwbare visuele marker in paraffineblokken en secties. 

Video: SpOT the Correct Tissue Every Time in Multi-tissue Blocks

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

Medicine

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&#38;E) annotations are used to target small tumor areas using a blade that is 250 &#181;m2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 &#181;m in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...

Cancer Research

Manual Construction of a Tissue Microarray using the Tape Method and a Handheld Microarrayer

The tissue microarray (TMA) is an important research tool in which many formalin fixed paraffin embedded (FFPE) samples can be represented in a single paraffin block. This is achieved by using tissue cores extracted from the region of interest of different donor FFPE blocks and arranging them into a single TMA paraffin block. Once constructed, sections from the completed TMA can be used to perform immunohistochemistry, chromogenic, fluorescence in situ hybridization (FISH) and RNA ISH studies to assess protein expression as well as genomic and transcriptional alterations in many samples simultaneously, thus minimizing tissue usage and reducing reagent costs. There are several different TMA construction techniques. One of the most common construction methods is the recipient method, which works best with cores of the same length for which a minimum length of 4 mm is recommended. Unfortunately, tissue blocks can be heavily resected during the diagnostic process, frequently resulting in "non-ideal" donor block thicknesses of less than 4 mm. The current article and video focus on the double-sided adhesive tape method; an alternative manual, low cost, easy to use, and rapid method to construct low density (&lt;50 cores) TMAs that is highly compatible with these non-ideal donor blocks. This protocol provides a step-by-step guide on how to construct a TMA using this method, with a focus on the critical importance of pathological review and post construction validation.

This protocol outlines the tape method on how to manually construct a tissue microarray using FFPE donor blocks of differing depths.

The tissue microarray (TMA) is an important research tool in which many formalin fixed paraffin embedded (FFPE) samples can be represented in a single ...

Mapping the Tumor Immune Landscape: An In-Depth Multiplex IHC Analysis Protocol

Multiplex Immunohistochemical Analysis of the Spatial Immune Cell Landscape of the Tumor Microenvironment

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. Multiplex immunohistochemistry is a valuable tool to visualize and identify different types of immune cells in tumor tissues while retaining its spatial information. Here we provide detailed protocols to analyze lymphocyte, myeloid, and dendritic cell populations in tissue sections. Starting from cutting formalin-fixed paraffin-embedded sections, automatic multiplex staining procedures on an automated platform, scanning of the slides on a multispectral imaging microscope, to the analysis of images using an in-house-developed machine learning algorithm ImmuNet. These protocols can be applied to a variety of tumor specimens by simply switching tumor markers to analyze immune cells in different compartments of the sample (tumor versus invasive margin) and apply nearest-neighbor analysis. This analysis is not limited to tumor samples but can also be applied to other (non-)pathogenic tissues. Improvements to the equipment and workflow over the past few years have significantly shortened throughput times, which facilitates the future application of this procedure in the diagnostic setting.

Author Spotlight: Unlocking Insights into the Immune Cell Landscape of Tumors

This protocol describes in detail how immune cell characterization of the tumor microenvironment using multiplex immunohistochemistry is carried out.

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. ...

Deciphering Tumor Microenvironment Using mIHC in Lung Squamous Carcinoma

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been considered the leading cause of death in lung cancer patients. The complexity of the tumor microenvironment requires effective methods to understand cell-to-cell relationships in tumor tissues. The multiplex immunohistochemistry (mIHC) technique has become a key tool for inferring the relationship between the expression of proteins upstream and downstream of signaling pathways in tumor tissues and developing clinical diagnoses and treatment plans. mIHC is a multi-label immunofluorescence staining method based on Tyramine Signal Amplification (TSA) technology, which can simultaneously detect multiple target molecules on the same tissue section sample to achieve different protein co-expression and co-localization analysis. In this experimental protocol, paraffin-embedded tissue sections of lung squamous carcinoma of clinical origin were subjected to multiplex immunohistochemical staining. By optimizing the experimental protocol, multiplex immunohistochemical staining of labeled target cells and proteins was achieved, solving the problem of autofluorescence and channel crosstalk in lung tissues. In addition, multiplex immunohistochemical staining is widely used in the experimental validation of tumor-related, high-throughput sequencing, including single-cell sequencing, proteomics, and tissue space sequencing, providing intuitive and visual pathology validation results.

Author Spotlight: Enhancing Multicolor Fluorescence Localization in Lung Carcinoma Sample

This experimental protocol describes and optimizes a multiplex immunohistochemistry (IHC) staining method, mainly by optimizing single-channel antibody incubation conditions and adjusting the settings of antibodies and channels to solve the problems of autofluorescence and channel crosstalk in lung cancer tissues of clinical origin.

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been ...

Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts 1 2. Approximately 250 consecutive sections (4 &mu;m thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (<a href="http://www.proteinatlas.org" target="_blank">www.proteinatlas.org</a>) (Figure 1) 3 4. The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins encoded by the human genome.

Tissue microarrays allows for an efficient method to gain concurrent information from a multitude of tissues. Representative parts of tissues are assembled into a single paraffin block. Sections from the block are used for immunohistochemistry and analysis of protein expression patterns. Digital scanning generates corresponding images for distribution of data.

The  tissue microarray (TMA) technology provides the means for high-throughput  analysis of multiple tissues and cells. The technique is used within the ...

Highly-Multiplexed Tissue Imaging with Raman Dyes

Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. Hence, highly multiplexed imaging technologies have been increasingly appreciated. Here, we describe an emerging platform of highly-multiplexed vibrational imaging of specific proteins with comparable sensitivity to standard immunofluorescence via electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes. This method circumvents the limit of spectrally-resolvable channels in conventional immunofluorescence and provides a one-shot optical approach to interrogate multiple markers in tissues with subcellular resolution. It is generally compatible with standard tissue preparations, including paraformaldehyde-fixed tissues, frozen tissues, and formalin-fixed paraffin-embedded (FFPE) human tissues. We envisage this platform will provide a more comprehensive picture of protein interactions of biological specimens, particularly for thick intact tissues. This protocol provides the workflow from antibody preparation to tissue sample staining, to SRS microscope assembly, to epr-SRS tissue imaging.

Electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes is a new platform for highly multiplexed epitope-based protein imaging. Here, we present a practical guide including antibody preparation, tissue sample staining, SRS microscope assembly, and epr-SRS tissue imaging.

Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. ...

Bioengineering

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

Immunology and Infection

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

SpOT the Correct Tissue Every Time in Multi-tissue Blocks

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