Prestige Antibodies Powered by Atlas Antibodies in Immunohistochemistry

Immunohistochemistry and the Human Protein Atlas

Immunohistochemistry (IHC) is the most widely used technique in histopathological diagnosis and research for detection of proteins in tissues and cells. Today, IHC can be applied in a high-throughput fashion for studying proteins by the use of Tissue Microarrays (TMAs). In the Human Protein Atlas Project, Prestige Antibodies have been used to analyze all human proteins using IHC and TMAs.^1,2 All resulting tissue and cell images are publicly available on the Human Protein Atlas web portal (www.proteinatlas.org).^3,4 In total, more than 500 high resolution IHC images from human tissue samples are presented for each Prestige Antibody. Each year protein expression and localization data of ~2,500 new proteins are added to the portal. By the end of 2015, a first draft of the localization of the full human proteome will be available.

Tissue Microarrays

The TMA technology provides an automated array-based high-throughput technique in which as many as 1,000 paraffin-embedded tissue samples can be brought into one paraffin block in an array format. This allows for protein expression profiling on a large scale. Each antibody in the Human Protein Atlas Project generates more than 500 high-resolution images corresponding to normal and cancer tissues. In this manner, an atlas for tissue expression and localization is built up for each protein with an available specific antibody. TMAs used within the Human Protein Atlas Project include samples from 48 different human normal tissue types and 20 different types of cancer. Normal tissues are sampled from 144 different individuals and cancer tissues are derived from 216 unique tumors.^1,2

TMAs are constructed by extracting cylinders of formalin fixed, paraffin embedded tissue from donor blocks with a sharp punch and assembling them into a recipient block with properly sized holes in a grid pattern (Figure 1). From one array block, ~250 sections can be achieved and prepared for IHC analysis.

Figure 1: Cylinders from donor blocks are extracted and inserted into a recipient block. A) Donor blocks of formalin fixed, paraffin embedded human tissues. B) Recipient block (Tissue Microarray) representing 48 different human normal tissue types ready to be sectioned and used for IHC analysis.

IHC Method in the Human Protein Atlas Project

Within the Human Protein Atlas Project, antibody production and analysis are performed in a high-throughput fashion.^3,4 Therefore, the immunohistochemistry procedure is highly automated and performed under standardized conditions. Antigen retrieval, Heat Induced Epitope Retrieveal (HIER), is performed in citrate buffer at pH 6, using a pressure boiler. The Prestige Antibodies are diluted using a dilution robot and staining is performed in an autostainer. A Horse Radish Peroxidase (HRP) - conjugated polymer together with the chromogen 3,3’ - diaminobenzidine (DAB) are used for detection. The specific binding of an antibody to its corresponding antigen results in a brown staining (Figure 2). The tissue section is counterstained with hematoxylin. Hematoxylin staining is unspecific and results in a blue coloring of both cells and extracellular material.

Figure 2. Schematic figure of the immunohistochemical staining reaction. Prestige Antibodies are used as primary antibodies and the secondary antibody is labeled with the enzyme HRP. HRP forms a complex with the substrate H₂O₂ and in the presence of the chromogen DAB, a brown color can be visualized using light microscopy. The signal can be amplified using an enzyme-linked dextran polymer (figure to the right).

Trained professionals determine the optimal dilution and approve antibodies based on a comparison of staining pattern, available information from gene and protein public databases, as well as in-house technical validation such as protein arrays and Western blots.

Scanning and Annotation

All immunostained slides are scanned to generate high-quality images. More than 7 terabyte of image data is generated each month. The images representing immunostained tissue sections (TMAs) are analyzed and annotated by certified pathologists using a web based annotation software. All images and annotations are published and freely available at the Human Protein Atlas portal (www.proteinatlas.org).

Subcellular Analysis Using Immunohistochemistry

Immunofluorescence-based imaging, with its advantage of high resolution and sensitivity, remains an established golden standard for visualization of proteins at a subcellular level. In addition, immunofluorescence allows the use of several different antibodies tagged with different fluorophores simultaneously, which enables a more detailed analysis of subcellular localization patterns. Data on where proteins are localized within a cell provides important information as to what basic functions a protein may have as well as a possibility to map possible other interacting proteins.

A vast majority of studies based on immunofluorescence are performed on cultured cells with the disadvantage of not being able to analyze cells in their natural tissue context. In addition to the subcellular protein profiling available through immunofluorescence, localization information at a subcellular level can also be achieved using Prestige Antibodies in immunohistochemistry (Figure 3). Figures 3 A-C show examples of immunohistochemical stainings using Prestige Antibodies for recognition of cell membrane-related proteins, Figures 3 D-F show examples of proteins expressed in different cytoplasmic compartments and Figures 3 G-I show proteins expressed in different nuclear structures.

Figure 3. Immunohistochemical stainings showing subcellular localization using Prestige Antibodies. Recognition of target antigen is represented by brown color.

Summary

• The use of Prestige Antibodies in Immunohistochemistry on Tissue Microarrays (TMAs) has allowed for protein expression profiling in a large-scale format.
• In the Human Protein Atlas Project, TMAs including samples from 48 different human normal tissue types and 20 different types of cancer are used for protein localization analysis.
• For each Prestige Antibody, more than 500 IHC images are publicly available on the Human Protein Atlas web portal www.proteinatlas.org.
• Each month The Human Protein Atlas Project produces 30 TMAs, performs IHC-analysis on 600 new antibodies and manually annotates the resulting IHC images from 240 antibodies.
• By the use of Prestige Antibodies in Immunohistochemistry studies, information on a subcellular level can be achieved.

About The Human Protein Atlas

The Human Protein Atlas is a public web portal managed by an academic project that aims to map the human proteome in a period of 10 years. More than 700 IHC, ICC, WB and IF images are presented for each antibody against human targets.

References

1. Pontén F, Jirström K, Uhlén M. The Human Protein Atlas - a tool for pathology. J Pathology 2008 216(4):387-93.
2. Kampf C, Andersson A-C, Wester K, Björling E, Uhlen M, Ponten F Antibody-based tissue profiling as a tool in clinical proteomics. Clin. Proteomics 2004 1(3-4):285-300.
3. Uhlén M, Björling E, Agaton C, Szigyarto CA, Amini B, Angelidou P, Asplund A, Berglund L, Bergström K, Hansson M, Hober S, Kampf C, Pontén F, et al. A Human Protein Atlas for Normal and Cancer Tissues Based on Antibody Proteomics. Mol Cell Proteomics 2005 4(12):1920-1932.
4. Berglund L, Björling E, Oksvold P, Fagerberg L, Asplund A, Wernérus H, Nilsson P, Lundberg E, Sivertsson A, Navani S, Wester K, Kampf C, Hober S, Pontén F, Uhlén M, et al. A gene-centric human protein atlas for expression profiles based on antibodies. Molecular & Cellular Proteomics 2008 7:2019-2027.

Learn more about Prestige Antibodies at sigma.com/prestige.