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 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...

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

Research

JoVE Journal

Biology

11.7K 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.

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

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...