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% powe...

<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. 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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>

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 le...

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>HistogelTM Specimen Processing Gel</td>
			<td>Thermo Scientific</td>
			<td>HG-4000-012</td>
			<td><a href="http://www.thermoscientific.com/en/product/richard-allan-scientific-histogel-specimen-processing-gel.html">http://www.thermoscientific.com/en/product/richard-allan-scientific-histogel-specimen-processing-gel.html&#160;</a></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 t...

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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 ...

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11.6K Views.  Frederick National Laboratory for Cancer Research. 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.