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. 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>

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

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11.6K vues.  Frederick National Laboratory for Cancer Research.  Le but de l'échantillon Orientation Tag (spot) est de fonctionner comme un outil d'orientation pour aider à l'identification de tissus individu dans des blocs de paraffine multi-tissus. Ces protocoles démontrent comment il est construit à partir de matériaux facilement communes d'histologie à faible coût et sert de repère visuel fiable dans des blocs de paraffine et des sections. 

Vidéo: 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...

manual construction of a tissue microarray using the tape method and a handheld microarrayer

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

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

a rapid filter insert-based 3d culture system for primary prostate cell differentiation

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

Here, we present a method for the establishment of a rapid in vitro system that supports the three dimensional culturing and subsequent luminal differentiation of primary prostate epithelial...

cryopreservation of cortical tissue blocks for the generation of highly enriched neuronal cultures

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures.  This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell...

monitoring cancer cell invasion and t-cell cytotoxicity in 3d culture

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated...

the multi-organ chip - a microfluidic platform for long-term multi-tissue coculture

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media...

real-time void spot assay

Real-Time Void Spot Assay

This article describes a new method to study mouse voiding behavior by incorporating video monitoring in the conventional void spot assay. This approach provides temporal, spatial, and volumetric information on the voiding events and details of mouse behavior during the light and dark phases of the...

maintaining and assessing various tissue and cell types of the eye using a novel pumpless fluidics system

Author Spotlight: Advancing Eye Physiology Research via a Multi-Channel Flow Culture for Optimal Tissue Maintenance and Real-Time Assessment 

Real-time analysis of live tissue yields important functional and mechanistic data. This paper describes the protocols and critical variables to ensure accurate and reproducible generation of data by a novel and pump-free multi-channel fluidics system that maintains and assesses a wide range of tissue and cell models.

Pumpless Multi-Channel Flow Culture to Maintain and Assess Tissues and Cells

multi-electrode array recordings of human epileptic postoperative cortical tissue

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in...

measuring real-time drug response in organotypic tumor tissue slices

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo...

capturing tissue repair in zebrafish larvae with time-lapse brightfield stereomicroscopy

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse...

setting up the pump-free multi-channel fluidic system and loading the tissue for culturing

Setting Up the Pump-Free Multi-Channel Fluidic System and Loading the Tissue for Culturing  

tissue engineering by intrinsic vascularization in an in vivo tissue engineering chamber

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

an intra-tissue radiometry microprobe for measuring radiance in situ in living tissue

An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue

In this paper, a method for measuring radiance in situ in living tissue is described. This work includes details of the construction of micro-scale probes for different measurements of radiance and irradiance, provides guidance for mounting tissue for the characterization of radiance, and outlines computational methods for analyzing the resulting data.

An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue

using an in vitro tissue perfusion system to detect the functional activities of isolated intestinal tubes in real time

Author Spotlight: Reliable and Reproducible In Vitro Assessment of Drug Impact on Rat Intestinal Tubes

We present a method for isolating rat intestinal tubes and assessing the impact of drugs on their tension, frequency, and amplitude in vitro. This method offers a valuable approach for researchers investigating intestinal...

Using An In Vitro Tissue Perfusion System to Detect the Functional Activities of Isolated Intestinal Tubes in Real Time

implantation of engineered tissue in the rat heart

Implantation of Engineered Tissue in the Rat Heart

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

tissue engineering of the intestine in a murine model

Tissue Engineering of the Intestine in a Murine Model

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold...

tracking morphogenetic tissue deformations in the early chick embryo

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three...

visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

The interrenal gland in zebrafish is the teleostean counterpart of the mammalian adrenal gland. This protocol introduces how to perform the 3-&#946;-hydroxysteroid dehydrogenase (&#916;5-4 isomerase; 3&#946;-Hsd) enzymatic activity assay, which detects differentiated steroidogenic cells in the developing...

multi-photon time lapse imaging to visualize development in real-time: visualization of migrating neural crest cells in zebrafish embryos

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish...

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 ...

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), ...

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

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 &amp;#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) ...

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 ...

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 ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...