This work was supported by grants from the Knut and Alice Wallenberg foundation. The entire staffs of the Human Protein Atlas centers in Uppsala, Stockholm and India are acknowledged for their efforts to generate the Human Protein Atlas. The authors wish to especially thank Frank Hammar and Sofie Gustafsson for assistance with the photos and Elen&eacute; Karlberg for assistance with the TMA production when filming.

1. How to Prepare Tissues for Tissue Microarray Production (Animation 1)
<ol>
 <li>Select relevant formalin fixed paraffin embedded material (tissue or cell samples) including corresponding hematoxylin stained tissue section.</li>
 <li>Mark relevant area on the tissue section. It is recommended to have a freshly cut hematoxylin stained section corresponding to the paraffin block.</li>
 <li>Design the template used for TMA production. Randomize the samples within the template to avoid artifacts caused by technical problems such as coverage of the antibody over the whole TMA section and sectioning problems. Add additional orientation markers to the template for orientation.</li>
 <li>Organize the tissues according to the template.</li>
</ol>
2. Manual Tissue Microarray Production (Essential for filming)
<ol>
<li> To be able to obtain a standardized length of the tissue cores, mark the stylet eg 4.5 mm (Figure 2). Guidelines for spacing between sample centers: 
core size 0.6 mm &#x2013; 0.8-1.0 mm 
core size 1.0 mm &#x2013; 1.8-2.0 mm 
core size 1.5 mm &#x2013; 2.0-3.0 mm 
core size 2.0 mm &#x2013; 2.5-4.0 mm 
It is recommended to leave 2.5-3.0 mm margins on each side of the paraffin block to avoid cracking of the paraffin. 
</li>
<li>
 <ol class="lalpha">
 <li> Mount the selected punches to be used. Start by loosening the hex socket screws that holds the punch in place. The punch is correctly positioned when the groove in the punch hub is firmly placed against the metal rod in the plastic v-block. Fasten the screws and make sure that the edge of the metal clip is horizontal.</li>
 <li> The recipient punch (marked in green and red, specific for distributor) should be placed to the left. The donor punch (marked in green and blue, specific for distributor), which has a slightly larger diameter, should be placed to the right (Figure 3).</li>
 </ol>
</li>
<li>
 <ol class="lalpha">
 <li> Move the punches along the x- and y-axes by using the XY adjustment knobs. The right adjustment knob moves the punches along the x-axis and the left adjustment knob moves the punches along the y-axis. 
 There is a digital micrometer readout on both adjustment knobs. These are activated when moving the knobs. </li>
 <li> Reset &rarr; press the ZERO/ABS button.
 
 Shift between inches and mm &rarr; press the IN/mm button.
 
 The space between the holes should be 2.0 mm (measured between the centers of the holes) when using a 1 mm punch for a 9x8 template.</li>
 </ol>
</li>
<li>
 <ol class="lalpha">
 <li> To ensure that you do not push the punch too far down into the block, hitting the cassette and destroying the punch, put a cassette without paraffin in the holder and set the bottom position of the recipient punch to 1 mm above the bottom of the cassette by using the depth stop screw at the upper left corner of the Z-guide (Figure 3). </li>
 <li> Put the recipient block in the holder and use the smallest screwdriver to fasten the screws. Do not fasten the screws to tight or the paraffin might break loose from the cassette. </li>
 <li> Push the recipient punch downward approximately 5 mm into the recipient block and use the handle in the punch to rotate the punch back and forth once (Figure 3). The hole will be 5 mm deep when pushing the punch as far down into the block as possible. </li>
 <li> Relieve the downward pushing pressure slowly, so that the springs can move the punch up. Use the stylet to empty the punch and discard the paraffin core. </li>
 </ol>
</li>
<li> Move the turret to switch to the donor punch (Figure 3). </li>
<li> Place the donor block bridge with the donor block over the recipient block holder (Figure 3). </li>
<li>
 <ol class="lalpha">
 <li> Push the donor punch into the selected area of the donor block. Use a corresponding hematoxylin and eosin stained slide where the region of interest has been marked to locate the tissue that is to be sampled. Manually hold the donor block in position with your left hand and push the punch with your right hand. Note that you should not push on the stylet. The depth stop does not block the punch motion at the proper position for the donor block, so it is important that you stop pushing when the second mark is visible on the stylet.</li>
 <li> Rotate the handle on the punch once (Figure 3). Slowly release the downwards pressure while still holding the donor block on the donor block bridge.</li>
 <li> The donor tissue is preferably punched 0.5 mm shorter than the recipient core. This process will ensure an adjustable alignment of the cores on the recipient block surface, avoiding loss of valuable representative tissue.</li>
 </ol>
</li>
<li> Remove the bridge and the donor block. Make sure that the donor punch is aligned with the hole that was made earlier (point 4). You must adjust the punch position using the set screws if the donor punch and the hole in the recipient block are not aligned. </li>
<li> Carefully push the punch downward until its tip reaches the top of the hole in the recipient block. While holding this position, use the stylet to empty the tissue core into the hole. The core surface should be in alignment with the block surface. </li>
<li> Move the turret back to the recipient punch. </li>
<li> Move to the next position with the XY adjustment knobs. </li>
<li> Repeat from point 4c above until the array is complete. </li>
<li> When the whole recipient block is finished, loosen the screws in the recipient block holder. Bake the block in 42 &deg;C for 40 minutes. Put the block with the sectioning area facing down on a glass slide. Place the glass slide with the block on top of a test tube holder (or other appropriate construction) and place in the oven. After baking, remove the test tube holder from the oven and flatten the surface of the block by gently stroking the glass slide. </li>
<li> Place the block with the glass slide on a cooling plate for 10 minutes.
 
 To ensure representative tissue throughout the TMA a quality control on each 50th section is performed.</li>
</ol>
3. How to Section TMAs (Animation 2)
<ol>
 <li>Cut 4 &mu;m thick sections by using a microtome with section transfer system (HM 355S, Microm). The system can be used with all current rotary microtomes. The system consists of a blade carrier with integrated transfer bridge, a heated water bath and a control unit. From the blade the sections glide on the wet transfer surface via a transfer bridge into the water bath.</li>
 <li>The sections should be taken from the water bath immediately after they have stretched out, to avoid melting of sensitive tissue spots (eg. lipid rich tissues like breast and brain). Place the section on a superfrost glass slide. The time the sections can be left in the water depends on the type of paraffin waxed used and the water temperature. Our lab use paraffin wax from HistoLab Products AB, which have a melting point of 56-58 &deg; C and we recommend a water bath temperature of 37-39 &deg;C.</li>
 <li>Place the slides in a slide holder for drying in room temperature (RT) over night.</li>
 <li>The slides are baked in 50 &deg;C for 12-24 hours. Using a microtom without STS you have to transport the section from the blade to a water bath manually by using e.g. a brush.</li>
</ol>
4. Test and Titration Procedure for Immunohistochemistry (Animation 3)
Each antibody is subjected to a standardized test procedure using specially designed TMAs containing a mix of tissues and different cell types. The aim is to assess an individual and optimized immunostaining protocol for each antibody. Initially one dilution, based on antibody stock concentration of the primary antibody is tested. The outcome of this test staining is used to guide further dilutions and optimization of the protocol.
<ol>
 <li>Deparaffinization and hydration in xylene and graded ethanol to distilled water are performed prior to immunostaining.</li>
 <li>A blocking step to quench endogeneous peroxidase is performed in 0.3 % H2O2 in 95% ethanol for 5 minutes.</li>
 <li>Heat Induced Epitope Retrieval (HIER) is performed in a retrieval buffer pH 6, using a pressure boiler (Decloaking chamber, Biocare Medical) as heat source for 4 minutes at 125 &deg;C. After completed boiling, slides remain in the pressure boiler and are allowed to cool to 90 &deg;C. The total processing time for HIER is approximately 45 minutes.</li>
 <li>If no conclusive result is obtained in the initial test, further tests are done based on the observed staining pattern 5 6. A pilot study, within the HPA high-throughput set-up, showed that the most useful antigen retrieval where HIER pH 6, but other retrieval methods such as pre-incubation with proteinase, microwave and boiling in buffer with higher or lower pH can of course be used. By changing the IHC condition eg changing antibody incubation times, antibody dilutions and Diaminobenzidine (DAB) incubation times the staining pattern can give more conclusive results.</li>
</ol>
5. Staining Program, Autostainer 480 (Thermo Fisher Scientific)
All incubations are done at RT and this protocol can also be used in as a manual setup with the same reagents.
<ol>
 <li>Rinse in wash buffer.</li>
 <li>Incubation with Ultra V block for 5 minutes.</li>
 <li>Rinse in wash buffer.</li>
 <li>Incubation with primary antibody for 30 minutes.</li>
 <li>Rinse in wash buffer (x2).</li>
 <li>Incubation with labeled horse radish peroxidase-polymer for 30 minutes.</li>
 <li>Rinse in wash buffer (x2).</li>
 <li>Developing in DAB solution for 10 minutes.</li>
 <li>Rinse in wash buffer (x2).</li>
 <li>Counterstaining in Mayers hematoxylin for 5 minutes*. When using a progressive staining method (e.g. Mayers hematoxylin) the intensity depends on time and no differentiation is required. A regressive staining method e.g. Harris hematoxylin requires differentiation to remove excess dye from tissues in order to accentuate a structure.</li>
 <li>Rinse in tap water*.</li>
 <li>Rinse in lithium carbonate water, diluted 1:5 from saturated solution for 1 minute*.</li>
 <li>Rinse in tap water for 5 minutes*.</li>
 <li>The slides are dehydrated in graded ethanol and slides are coverslipped (PERTEX, Histolab)</li>
</ol>
All reagents are applied at a volume of 300 &mu;l per slide. 
* Steps 10-14 are done in a histostaining instrument (Autostainer XL, Leica) and coverslipping is additionally done in a coverslipper system (Automated glass coverslipper CV5030, Leica).
6. How to Scan a Slide
The method of scanning slides is dependant on the digital scanner being utilized, this protocol only defines how to scan a slide using the automated scanning system Aperio XT (Aperio Technologies). In the Human Protein Atlas set-up 20x magnification for the tissue and 40x for the cells are used. Automated scanning could encounter focus problems due to heterogeneity of the tissue and the number of different tissues included in a TMA.
<ol>
 <li>Clean the glass slide from dirt, glue and check for air bubbles between the coverslip and the section. In case of air bubbles remount the slide.</li>
 <li>Put the slide in a rack and place the rack in the digital scanner. The time to scan a TMA at 20x magnification is approximately 6 minutes. A software from Aperio is used to select area of interest. Focus point selection is made automatically within the software. Manually selection of scan area and focus points is also possible.</li>
 <li>After the image has been generated it can be used for evaluation and annotation of the IHC staining. Images remain as a result file of the experiment and can be distributed to colleagues for discussions and further used for publishing the data. The images can be viewed in a freely available software from Aperio.</li>
</ol>
7. How to Search for your Favorite Protein in the Human Protein Atlas
<ol>
 <li>Browse to <a href="http://www.proteinatlas.org." target="_blank">www.proteinatlas.org. </a></li>
 <li>Enter the name of your favorite protein, Ensembl id, UniProt accession number or HGNC name eg insulin.</li>
 <li>Enter the summary page and scroll down to normal tissue and organ summary. The overview shows protein expression in pancreas.</li>
 <li>Click on "more tissue data". You enter the normal tissues view where you can see all tissues included sorted by organ.</li>
 <li>Click on pancreas to view the IHC results for 3 antibodies directed towards the same protein. To view a high-resolution image of pancreas click on the image.</li>
 <li>Additional information about protein expression in cancer tissue, cells and further validation data within the Human Protein Atlas can be found by browsing the Human Protein Atlas website.</li>
</ol>
8. Representative Results
Animation 1. How to prepare tissues for tissue microarray production. <a href="https://www.jove.com/files/ftp_upload/3620/3620_animation_1_Web.mov" target="_blank">Click here to view Animation 1</a>.
Animation 2. How to section TMAs. <a href="https://www.jove.com/files/ftp_upload/3620/3620_animation_2_Web.mov" target="_blank">Click here to view Animation 2</a>.
Animation 3. The immunohistochemistry technique is described. <a href="https://www.jove.com/files/ftp_upload/3620/3620_animation_3_Web.mov" target="_blank">Click here to view Animation 3</a>.
<img alt="Figure 1" src="/files/ftp_upload/3620/3620fig1.jpg" /> 
Figure 1. Overall scheme of the process within the Human Protein Atlas Uppsala.
<img alt="Figure 2" src="/files/ftp_upload/3620/3620fig2.jpg" /> 
Figure 2. Donor stylet marked for standardized length of tissue cores eg 4.5 mm.
<img alt="Figure 3" src="/files/ftp_upload/3620/3620fig3.jpg" /> 
Figure 3. Manual tissue microarrayer.
<img alt="Figure 4" src="/files/ftp_upload/3620/3620fig4.jpg" /> 
Figure 4. Examples of different quality of produced TMAs. (A) A straight and properly aligned TMA with sufficient space between the rows and columns as well as to the edge of the paraffin. (B) A TMA with some of the cores too close to the edge resulting in probable difficulties when sectioning. (C) A TMA that have been baked for too long resulting in a collapsed TMA without the correct template.
<img alt="Figure 5" src="/files/ftp_upload/3620/3620fig5.jpg" /> 
Figure 5. Hematoxylin and eosin stained TMAs. (A) A properly cut section with straight and aligned columns and rows. Using a bad or dirty blade could result in splitting (B) or compressed sections (C).
<img alt="Figure 6" src="/files/ftp_upload/3620/3620fig6.jpg" /> 
Figure 6. Titration of antibodies. (A) Reaction center cells in lymph node shows an optimally titrated antibody resulting in a specific cytoplasmic staining (shown in brown) without background. (B) The tissue section shows weak staining. Negative or weak staining is found when the concentration of primary antibody is too low or if the target protein is not present in the immunostained tissues. (C) The tissue section is overstained due to use of too high concentration of primary antibody. The dark color results in difficulties determining the subcellular location due to spill over, cross reactivity and false positivity.

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We have nothing to disclose.

Immunohistochemistry is by far the most commonly used application for TMAs. The combination of IHC and TMAs can be used in several different settings, e.g. high-throughput screening of protein expression patterns in tissues 7 8 and cells 9 10, biomarker discovery studies based on tissue samples from large patient cohorts 11 12 and more basic tumor biology studies, including different phenotypes/genotypes, differentiation stages, progression and metastasis 13. One advantage of using TMAs is that material from large cohorts can be analyzed at one time, both saving valuable biological material and ensuring more reproducible experiments. Moreover, the use of TMA technology saves on reagent costs and laboratory processing time. As a TMA only contains a limited amount of tissue, tissue heterogeneity is an issue. Depending on the type of tissue and questions to be addressed, several samples may be needed from the same specimen. For tumor tissues, the use of two to four cores from each specimen is recommended as it has been shown to result in a high degree of accuracy in representation of whole tissue sections 13 14. Depending on tissue composition the diameter of the punches (ranging from 0.6 mm to 2 mm) can be adjusted. Tissues are complex and composed of both several different cell types, structures and extra-cellular matrix. The composition of each different tissue type containing variable amount of fat, blood vessels, connective tissue, cartilage etc., will affect the pressure needed for punching and collecting separate cores. It is therefore of importance to practice all steps in the procedure on material that is not of any value, before producing a TMA with defined tissues for the intended experiments. 
When sectioning a variety of different tissues within one TMA block the composition of the block could influence the ability to acquire high quality sections. Certain tissues, e.g. skin, bone marrow, fat, brain and breast, render sectioning of the TMA block difficult due to the effect of differences in texture effecting how sections stretch out in the water bath and how different hardness is cut by the blade etc. It is therefore recommended to group tissues with similar features e.g. lipid rich tissue into one TMA, when applicable. The majority of cancer tissues are in this sense homogenous, which facilitates sectioning of cancer TMAs. An alternative sectioning method for stabilizing the microarray cores can be employed when handling a TMA with heterogeneous types of tissues. The use of an adhesive tape can be helpful to avoid the loss of cores and fall-out in the sectioned TMA. The use of adhesive tape should be employed with caution, since the tape can influence the thickness of sectioned cores and subsequently also the outcome of IHC staining 15. In the Human Protein Atlas set-up a TMA template of 9x8 (excluding markers) cores are used. It is of importance to obtain maximal number of sections and keep all tissues represented throughout the block. To save reagents and scanning time, 2 sections are placed on one glass slide allowing a maximum template of 9x8.
The major repository for protein information, Universal Protein Resource 16 includes approximately 20.300 reviewed human protein entries, of which only approximately 66% have evidence at the protein level. Also for a majority of the genes for which there is evidence of existence on the protein level, there is scarce or no information on protein function and distribution of expression. An important challenge for the future is to analyze the distribution pattern and relative abundance of these unknown proteins in various types of human tissue. The information from databases such as Uniprot, ENSEMBL, published data from PubMed can be used as a guide to establish if immunohistochemical staining patterns using antibodies towards unknown proteins represent actual protein profiles of the intended target protein. In addition, several other validation strategies are needed to ensure antibody functionality, for example antibody functionality in protein arrays, Western blot, immunofluorescence, immuno-precipitation and pull-down experiments. Perhaps the most important tool for validation of antibody functionality is to use paired antibodies, i.e. two different antibodies raised against separate, non-overlapping epitopes on the same target protein, to compare assay-specific outcome 17.
Based on the compiled information, antibodies are tested and titrated according to IHC standards and the experience from testing and validating over 35.000 antibodies in the Human Protein Atlas project. For over 20% of all genes with protein profiles, data from paired antibodies (2 or more antibodies towards non-overlapping epitopes on the same protein) has been used to determine a best estimate of the corresponding protein expression pattern. Paired antibodies provide an ultimate strategy for validating specific immunohistochemistry-based protein expression patterns. This strategy is valuable because the multitude of different tissues included in the basic screening increases the risk for cross-reactivity. It should also be noted that certain tissues are in general more prone to show background staining e.g macrophages, smooth muscle, distal tubules in kidney and hepatocytes in liver. Additional issues to consider include variations in tissue processing techniques over time for the tissues taken from archival material, which can result in false negative or inappropriate positive IHC staining patterns. This phenomenon is also encountered in large section analysis. Both negative and positive controls are vital and should be used whenever possible. For deeper studies including functional analyses, cell lines with forced expression and specific knock-down of corresponding transcripts (siRNA technology) can be used to create controls. For optimal results in IHC, it is important to test and use antigen retrieval systems, since the formalin fixation induces various chemical modifications e.g. cross-linking, hydrolysis of Schiff bases masking the antigen 18.
In conclusion, antibody-based proteomics employing TMA technology and IHC is a powerful strategy for generating protein expression data on a large scale. The Human Protein Atlas project has been set up to create a map of human protein expression in normal human tissues and cancer. The aim of this project is to present a first draft of a comprehensive Human Protein Atlas by 2015. In addition to providing protein profiles in normal organs and tissues, this effort also provides a starting point for biomedical research projects, including clinical biomarker discovery efforts. The ultimate goal is to add a next information layer on top of the human genome sequence that will be crucial for a deeper understanding of the biology underlying various disease states, and provide a basis for developing novel diagnostic and therapeutic tools.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Wash buffer</td>
 <td>Thermo Fisher Scientific</td>
 <td>TA-999-TT</td>
 </tr>
 <tr>
 <td>Citrate buffer pH6</td>
 <td>Thermo Fisher Scientific</td>
 <td>TA-250-Pm1X</td>
 </tr>
 <tr>
 <td>Antibody diluent</td>
 <td>Thermo Fisher Scientific</td>
 <td>TA-125-UD</td>
 </tr>
 <tr>
 <td>UltraVision LP HRP polymer </td>
 <td>Thermo Fisher Scientific</td>
 <td>TL-125-HL</td>
 </tr>
 <tr>
 <td>DAB plus substrate system </td>
 <td>Thermo Fisher Scientific</td>
 <td>TA-125-HDX</td>
 </tr>
 <tr>
 <td>Ultra V Block</td>
 <td>Thermo Fisher Scientific</td>
 <td>TA-125-UB</td>
 </tr>
 <tr>
 <td>Primary Antibody Enhancer</td>
 <td>Thermo Fisher Scientific</td>
 <td>TL-125-PB</td>
 </tr>
 <tr>
 <td>Mayers hematoxylin</td>
 <td>Histolab</td>
 <td>01820</td>
 </tr>
 <tr>
 <td>PERTEX</td>
 <td>Histolab</td>
 <td>00871.0500</td>
 </tr>
 <tr>
 <td colspan="3">&nbsp;</td>
 </tr>
 <tr>
 <td>Name of the equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Manual tissue micro arrayer</td>
 <td>Estigen, Beecher Instrument</td>
 <td>MTA-1</td>
 </tr>
 <tr>
 <td>Waterfall microtome</td>
 <td>Thermo Fisher Scientific</td>
 <td>Microm HM 355S</td>
 </tr>
 <tr>
 <td>Automated image scanner</td>
 <td>Aperio Technologies</td>
 <td>XT</td>
 </tr>
 <tr>
 <td>Automated slide staining system</td>
 <td>Thermo Fisher Scientific</td>
 <td>Autostainer 480S-2D</td>
 </tr>
 <tr>
 <td>Automated slide staining system for deparafinization and dehydration</td>
 <td>Leica Biosystems</td>
 <td>Autostainer XL</td>
 </tr>
 <tr>
 <td>Automated glass coverslipper</td>
 <td>Leica Biosystems</td>
 <td>CV5030</td>
 </tr>
 <tr>
 <td>Decloaking chamber</td>
 <td>Biocare Medical</td>
 <td>DC2008INTEL</td>
 </tr>
 </tbody>
</table>

production of tissue microarrays, immunohistochemistry staining and digitalization within the human protein atlas

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

Watch this Scientific Journal Video about Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas at JoVE.com

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

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

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

production-tissue-microarrays-immunohistochemistry-staining

Research

JoVE Journal

Biology

55.8K vistas.  Uppsala University. Microarrays de tejidos permite un método eficaz para obtener información simultánea de una multitud de tejidos. Piezas representativas de los tejidos se integran en un único bloque de parafina. Las secciones del bloque se utilizan para inmunohistoquímica y el análisis de patrones de expresión de proteínas. Exploración digital genera imágenes correspondientes para la distribución de datos.

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

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 ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic bacteria of special relevance for a wide number of hosts. P. syringae enters the plant from the leaf surface and multiplies rapidly within the apoplast, forming microcolonies that occupy the intercellular space. The constitutive expression of fluorescent proteins by the bacteria allows for visualization of the microcolonies and monitoring of the development of the infection at the microscopic level. Recent advances in single-cell analysis have revealed the large complexity reached by clonal isogenic bacterial populations. This complexity, referred to as phenotypic heterogeneity, is the consequence of cell-to-cell differences in gene expression (not linked to genetic differences) among the bacterial community. To analyze the expression of individual loci at the single-cell level, transcriptional fusions to fluorescent proteins have been widely used. Under stress conditions, such as those occurring during colonization of the plant apoplast, P. syringae differentiates into distinct subpopulations based on the heterogeneous expression of key virulence genes (i.e., the Hrp type III secretion system). However, single-cell analysis of any given P. syringae population recovered from plant tissue is challenging due to the cellular debris released during the mechanical disruption intrinsic to the inoculation and bacterial extraction processes. The present report details a method developed to monitor the expression of P. syringae genes of interest at the single-cell level during the colonization of Arabidopsis and bean plants. The preparation of the plants and the bacterial suspensions used for inoculation using a vacuum chamber are described. The recovery of endophytic bacteria from infected leaves by apoplastic fluid extraction is also explained here. Both the bacterial inoculation and bacterial extraction methods are empirically optimized to minimize plant and bacterial cell damage, resulting in bacterial preparations optimal for microscopy and flow cytometry analysis.

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

The present protocol describes a method that allows single-cell gene expression analysis on Pseudomonas syringae populations grown within the plant apoplast.

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic ...