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 – 0.8-1.0 mm 
core size 1.0 mm – 1.8-2.0 mm 
core size 1.5 mm – 2.0-3.0 mm 
core size 2.0 mm – 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 → press the ZERO/ABS button.
 
 Shift between inches and mm → 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 °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 μm 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 ° C and we recommend a water bath temperature of 37-39 °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 °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 °C. After completed boiling, slides remain in the pressure boiler and are allowed to cool to 90 °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 μl 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="Tissue microarray (TMA) analysis diagram showing antibody staining and digital slide processing." src="/files/ftp_upload/3620/3620fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Overall scheme of the process within the Human Protein Atlas Uppsala.
<img alt="Capacitor measurement, diagram showing cylindrical dimensions with labeled 4.5 mm rod length." src="/files/ftp_upload/3620/3620fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Donor stylet marked for standardized length of tissue cores eg 4.5 mm.
<img alt="Tissue microarray instrument with stylet and turret for sample preparation in microscopy analysis." src="/files/ftp_upload/3620/3620fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Manual tissue microarrayer.
<img alt="Static equilibrium analysis, chromatography result diagram, protein separation spots on gel pads." src="/files/ftp_upload/3620/3620fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
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="Histology staining result on slides; protein detection method showing intensity variations." src="/files/ftp_upload/3620/3620fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
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="Immunohistochemistry analysis of three tissue samples; comparative antibody binding results." src="/files/ftp_upload/3620/3620fig6.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
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.

<ol>
	<li>Battifora, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multitumor+(sausage)+tissue+block:+novel+method+for+immunohistochemical+antibody+testing.">The multitumor (sausage) tissue block: novel method for immunohistochemical antibody testing.</a> Lab Invest. 55, 244-244 (1986).</li><li>Kononen, J., Bubendorf, L., Kallioniemi, A. <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+for+high-throughput+molecular+profiling+of+tumor+specimens.">Tissue microarrays for high-throughput molecular profiling of tumor specimens.</a> Nat. Med. 4, 844-844 (1998).</li><li>Berglund, L., Bjorling, E., Oksvold, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genecentric+Human+Protein+Atlas+for+expression+profiles+based+on+antibodies.">A genecentric Human Protein Atlas for expression profiles based on antibodies.</a> Mol. Cell Proteomics. 7, 2019 (2008).</li><li>Uhlen, M., Bjorling, E., Agaton, 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+human+protein+atlas+for+normal+and+cancer+tissues+based+on+antibody+proteomics.">A human protein atlas for normal and cancer tissues based on antibody proteomics.</a> Mol. Cell Proteomics. 4, 1920 (2005).</li><li>Paavilainen, L., Edvinsson, A., Asplund, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+tissue+fixatives+on+morphology+and+antibody-based+protein+profiling+in+tissues+and+cells.">The impact of tissue fixatives on morphology and antibody-based protein profiling in tissues and cells.</a> J. Histochem. Cytochem. 58, 237-237 (2010).</li><li>Paavilainen, L., Wernerus, H., Nilsson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+monospecific+antibodies:+a+comparison+study+with+commercial+analogs+using+immunohistochemistry+on+tissue+microarrays.">Evaluation of monospecific antibodies: a comparison study with commercial analogs using immunohistochemistry on tissue microarrays.</a> Appl. Immunohistochem. Mol. Morphol. 16, 493-493 (2008).</li><li>Kampf, C., Andersson, A. C., Wester, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-based+tissue+profiling+as+a+tool+for+clinical+proteomics.">Antibody-based tissue profiling as a tool for clinical proteomics.</a> Clinical Proteomics. 1, 285-285 (2004).</li><li>Ponten, F., Jirstrom, K., Uhlen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Human+Protein+Atlas+-+a+tool+for+pathology.">The Human Protein Atlas - a tool for pathology.</a> J. Pathol. 216, 387-387 (2008).</li><li>Andersson, A. C., Stromberg, S., Backvall, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein+expression+in+cell+microarrays:+a+tool+for+antibody-based+proteomics.">Analysis of protein expression in cell microarrays: a tool for antibody-based proteomics.</a> J. Histochem. Cytochem. 54, 1413-14 (2006).</li><li>Stromberg, S., Bjorklund, M. G., Asplund, 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+high-throughput+strategy+for+protein+profiling+in+cell+microarrays+using+automated+image+analysis.">A high-throughput strategy for protein profiling in cell microarrays using automated image analysis.</a> Proteomics. 7, 2142 (2007).</li><li>Jogi, A., Brennan, D. J., Ryden, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+expression+of+the+RNA-binding+protein+RBM3+is+associated+with+an+improved+clinical+outcome+in+breast+cancer.">Nuclear expression of the RNA-binding protein RBM3 is associated with an improved clinical outcome in breast cancer.</a> Mod. Pathol. 22, 1564 (2009).</li><li>Magnusson, K., De Wit, M., Brennan, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SATB2+in+combination+with+cytokeratin+20+identifies+over+95%+of+all+colorectal+carcinomas.">SATB2 in combination with cytokeratin 20 identifies over 95% of all colorectal carcinomas.</a> Am. J. Surg. Pathol. 35, 937 (2011).</li><li>Nocito, A., Kononen, J., Kallioniemi, O. 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+microarrays+(TMAs)+for+high-throughput+molecular+pathology+research.">Tissue microarrays (TMAs) for high-throughput molecular pathology research.</a> Int. J. Cancer. 94, 1 (2001).</li><li>Rimm, D. L., Camp, R. L., Charette, L. A. <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+new+technology+for+amplification+of+tissue+resources.">Tissue microarray: a new technology for amplification of tissue resources.</a> Cancer J. 7, 24 (2001).</li><li>Catchpoole, D., Mackie, N., McIver, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tape+transfer+sectioning+of+tissue+microarrays+introduces+nonspecific+immunohistochemical+staining+artifacts.">Tape transfer sectioning of tissue microarrays introduces nonspecific immunohistochemical staining artifacts.</a> Biotech. Histochem. , (2010).</li><li>UniProt. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic acids research. 39, D214 (2011).</li><li>Uhlen, M., Oksvold, P., Fagerberg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+knowledge-based+Human+Protein+Atlas.">Towards a knowledge-based Human Protein Atlas.</a> Nature Biotechnol. 28, 1248 (2010).</li><li>Leong, A. S., Leong, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+in+immunohistology.">Standardization in immunohistology.</a> Method Mol. Cell Biol. 724, 37 (2011).</li></ol>

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

56.0K Views.  Uppsala University. Weefsel microarrays zorgt voor een efficiënte methode om gelijktijdige informatie te verkrijgen uit een veelheid van weefsels. Representatieve delen van weefsels worden samengevoegd tot een paraffineblok. Secties van het blok voor immunohistochemie en analyse van eiwit expressie. Digitaal scannen genereert corresponderende beelden voor de distributie van gegevens.

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

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Medicine

Digital Analysis of Immunostaining of ZW10 Interacting Protein in Human Lung Tissues

The purpose of this study is to introduce a methodology of the immunostaining of human lung tissues, followed by whole-slide digital scanning and image analysis. Digital scanning is a fast way to scan a stack of slides and produce digital images with high quality. It can produce concordant results with conventional light microscopy (CLM) by pathologists. Furthermore, the availability of digital images makes it possible that the same slide can be concurrently observed by multiple people. Moreover, digital images of slides can be stored in a database, which means the long-term deterioration of glass slides is avoided. The limitations of this technique are as follows. First, it needs high-quality prepared tissue and the original immunohistochemistry (IHC) slides without any damage or excess sealant residue. Second, tumor or nontumor areas should be specified by experienced pathologists before the analysis using software, in order to avoid any confusion about the tumor or nontumor areas during scoring. Third, the operator needs to control the color reproduction throughout the digitization process in whole-slide imaging.

ZW10 interacting protein (ZWINT) participates in the mitotic spindle checkpoint and the pathogenesis of carcinoma. Here, we introduce a methodology of the immunostaining of ZWINT in human lung cancer tissues, followed by the digital scanning of whole slides and image analysis. This methodology can provide high-quality digital images and reliable results.

The purpose of this study is to introduce a methodology of the immunostaining of human lung tissues, followed by whole-slide digital scanning and image ...

Cancer Research

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein and RNA multiplexing by leveraging photo-cleavable oligo-tagged antibodies and probes, respectively. Oligos are cleaved and quantified from specific regions across the tissue to elucidate the underlying biology. Here, the study demonstrates that automated custom antibody visualization protocols can be utilized to guide ROI selection in conjunction with spatial proteomics assays. This specific method did not show acceptable performance with spatial transcriptomics assays. The protocol describes the development of a 3-plex immunofluorescent (IF) assay for marker visualization on an automated platform, using tyramide signal amplification (TSA) to amplify the fluorescent signal from a given protein target and increase the antibody pool to choose from. The visualization protocol was automated using a thoroughly validated 3-plex assay to ensure quality and reproducibility. In addition, the exchange of DAPI for SYTO dyes was evaluated to allow imaging of TSA-based IF assays on the spatial profiling platform. Additionally, we tested the ability of selecting small ROIs using the spatial transcriptomics assay to allow the investigation of highly-specific areas of interest (e.g., areas enriched for a given cell type). ROIs of 50 &#181;m and 300 &#181;m diameter were collected, which corresponds to approximately 15 cells and 100 cells, respectively. Samples were made into libraries and sequenced to investigate the capability to detect signals from small ROIs and profile-specific regions of the tissue. We determined that spatial proteomics technologies highly benefit from automated, standardized protocols to guide ROI selection. While this automated visualization protocol was not compatible with spatial transcriptomics assays, we were able to test and confirm that specific cell populations can successfully be detected even in small ROIs with the standard manual visualization protocol.

Here, we describe a protocol for fine-tuning regions of interest (ROIs) for Spatial Omics technologies to better characterize the tumor microenvironment and identify specific cell populations. For proteomics assays, automated customized protocols can guide ROI selection, while transcriptomics assays can be fine-tuned utilizing ROIs as small as 50 &#181;m.

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein ...

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

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

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

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

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...

Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell populations. To characterize proteome-level variation and tumor heterogeneity at scale, high-throughput methods are needed to selectively isolate discrete cellular populations in solid tumor malignancies. This protocol describes a high-throughput workflow, enabled by artificial intelligence (AI), that segments images of hematoxylin and eosin (H&#38;E)-stained, thin tissue sections into pathology-confirmed regions of interest for selective harvest of histology-resolved cell populations using laser microdissection (LMD). This strategy includes a novel algorithm enabling the transfer of regions denoting cell populations of interest, annotated using digital image software, directly to laser microscopes, thus enabling more facile collections. Successful implementation of this workflow was performed, demonstrating the utility of this harmonized method to selectively harvest tumor cell populations from the TME for quantitative, multiplexed proteomic analysis by high-resolution mass spectrometry. This strategy fully integrates with routine histopathology review, leveraging digital image analysis to support enrichment of cellular populations of interest and is fully generalizable, enabling harmonized harvests of cell populations from the TME for multiomic analyses.

This protocol describes a high-throughput workflow for artificial intelligence-driven segmentation of pathology-confirmed regions of interest from stained, thin tissue section images for enrichment of histology-resolved cell populations using laser microdissection. This strategy includes a novel algorithm enabling the transfer of demarcations denoting cell populations of interest directly to laser microscopes.

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell ...

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

Multiplex Immunofluorescence and Spatial Image Analysis of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers.
We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types.
This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

Author Spotlight: Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment  

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the laboratory.

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...

A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into a &#8216;recipient&#8217; block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 &#8216;recipient&#8217; blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.

A Next-generation Tissue Microarray ngTMA Protocol for Biomarker Studies

The protocol aims at optimizing the construction and quality of tissue microarrays for biomarker research. It includes aspects of planning and design, digital pathology, virtual slide annotation, and automated tissue arraying.

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into ...

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

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

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

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

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

Samenvatting

Meer video's verkennen