Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University
Kampf, C., Olsson, I., Ryberg, U., Sjöstedt, E., Pontén, F. Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas. J. Vis. Exp. (63), e3620, doi:10.3791/3620 (2012).
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 μm 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 (www.proteinatlas.org) (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.
1. How to Prepare Tissues for Tissue Microarray Production (Animation 1)
2. Manual Tissue Microarray Production (Essential for filming)
3. How to Section TMAs (Animation 2)
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.
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.
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.
7. How to Search for your Favorite Protein in the Human Protein Atlas
8. Representative Results
Animation 1. How to prepare tissues for tissue microarray production. Click here to view Animation 1.
Animation 2. How to section TMAs. Click here to view Animation 2.
Animation 3. The immunohistochemistry technique is described. Click here to view Animation 3.
Figure 1. Overall scheme of the process within the Human Protein Atlas Uppsala.
Figure 2. Donor stylet marked for standardized length of tissue cores eg 4.5 mm.
Figure 3. Manual tissue microarrayer.
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.
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).
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.
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.
We have nothing to disclose.
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 Elené Karlberg for assistance with the TMA production when filming.
|Wash buffer||Thermo Fisher Scientific, Inc.||TA-999-TT|
|Citrate buffer pH6||Thermo Fisher Scientific, Inc.||TA-250-Pm1X|
|Antibody diluent||Thermo Fisher Scientific, Inc.||TA-125-UD|
|UltraVision LP HRP polymer||Thermo Fisher Scientific, Inc.||TL-125-HL|
|DAB plus substrate system||Thermo Fisher Scientific, Inc.||TA-125-HDX|
|Ultra V Block||Thermo Fisher Scientific, Inc.||TA-125-UB|
|Primary Antibody Enhancer||Thermo Fisher Scientific, Inc.||TL-125-PB|
|Manual tissue micro arrayer||Estigen, Beecher Instrument||MTA-1|
|Waterfall microtome||Thermo Fisher Scientific, Inc.||Microm HM 355S|
|Automated image scanner||Aperio Technologies||XT|
|Automated slide staining system||Thermo Fisher Scientific, Inc.||Autostainer 480S-2D|
|Automated slide staining system for deparafinization and dehydration||Leica Microsystems||Autostainer XL|
|Automated glass coverslipper||Leica Microsystems||CV5030|
|Decloaking chamber||Biocare Medical||DC2008INTEL|