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The protein atlas contains histological images of sections from human tissues. The images represent a view similar to what is seen in a microscope when examining sections of tissue on glass slides. Each antibody in the database has been used for immunohistochemical staining of both normal and cancer tissue. The specific binding of an antibody to its corresponding antigen results in a brown-black staining. The tissue section is counterstained with hematoxylin to enable visualization of microscopical features. Hematoxylin staining is unspecific and results in a blue coloring of both cells and extracellular material.
Tissue microarrays provides the possibility to immunohistochemically stain a large number and variety of normal and cancer tissues. The used tissue microarrays include samples from 48 different normal tissue types and 20 different types of cancer. For each antibody, protein expression patterns in normal tissue can be viewed as triplicate samples and in cancer tissue as duplicate samples. Normal tissues are sampled from 144 (48 x 3) different individuals and cancer tissues are derived from 216 (216 x 2) unique tumors. Normally, a fraction (<5%) of the 576 images are missing for each antibody due to technical issues. Samples of normal and cancer tissue have been collected from anonymized paraffin embedded material of surgical specimens, in accordance with approval from the local ethics
committee.
Since specimens are derived from surgical material, normal is here defined as non-neoplastic and morphologically normal. It is not always possible to obtain fully normal tissues and thus several of the tissues denoted as normal will include alterations due to inflammation, degeneration and tissue remodeling. In rare tissues, hyperplasia or benign proliferations are included as exceptions. It should also be noted that within normal morphology there exists inter-individual differences and variations due to primary diseases, age, sex etc. Such differences may also effect protein expression and thereby immunohistochemical staining patterns.
Samples from cancer are also derived from surgical material. The inclusion of tumors has been based on availability and representativity. Due to subgroups and heterogeneity of tumors within each cancer type, included cases represent a typical mix of specimens from surgical pathology. However, an effort has been made to include high and low grade malignancies where such is applicable. In certain tumor groups, subtypes have been included, e.g. breast cancer includes both ductal and lobular cancer, lung cancer includes both squamous cell carcinoma and adenocarcinoma and liver cancer includes both hepatocellular and cholangiocellular carcinoma etc. Tumor heterogenity and inter-individual differences is also reflected in diverse expression of proteins resulting in variable immunohistochemical staining patterns.
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As a complement to the representation of normal and cancer tissue, the protein atlas displays images of a selection of widely used and well characterized human cell lines as well as cell samples from normal individuals and leukemia/lymphoma patients.
A cell microarray has been used to enable immunohistochemical staining of a panel of cell lines and cell samples. Duplicates from 47 cell lines and 12 samples of primary blood cells renders a total of 118 cell images per antibody. Normally, a small fraction of the images are missing due to technical issues. Included cell lines are derived from DSMZ, ATCC or academic research groups (kindly provided by the founder of such cell lines). Information regarding sex and age of the donor, cellular origin and source is listed under the Protein Atlas Cell Line Dictionary. All cells were fixed in 4% paraformaldehyde prior to parafin embedding and immunohistochemical staining.
The cell microarray enables a wide representation of lymphoma/leukemia as well as other cell types that are difficult to include in a large scale atlas due to limited availability. Certain phenotypes not present in the tissue microarrays are also included, e.g. sarcoma, choriocarcinoma, small cell lung carcinoma. In addition, several solid tumors included in the tissue microarrays are represented by a cell line derived from corresponding tumor type. The simultaneous staining using the same protocol for both tissue and cell arrays allows for comparison of IHC staining between cell lines and tissue.
The immunohistochemical protocols used result in a brown-black staining, localized where an antibody has bound to its corresponding antigen. The section is furthermore histochemically counterstained with hematoxylin to enable visualization of microscopical features. Hematoxylin staining is unspecific, and results in a blue coloring of both cells and extracellular material.
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As a complement to the immunohistochemically stained cells and tissues, the protein atlas displays high resolution, multicolor images of immunofluorescently stained cells. This provides spatial information on protein expression patterns on a fine cellular and subcellular level.
Three cell lines, U-2OS, A-431 and U-251MG, originated from different human tissues have been chosen to be included in the immunofluorescent analysis. To enhance the probability for a large number of proteins to be expressed, we selected cell lines from different lineages, i.e. tumor cell lines from mesenchymal, epithelial and glial tumors. The selection was furthermore based on morphological characteristics, widespread use and multitude of publications using these cell lines. Information regarding sex and age of the donor, cellular origin and source is listed under the Protein Atlas Cell Line Dictionary.
Besides the HPA antibodies, the cells are also stained with cellular probes targeting specific compartments in the cells in order to facilitate the annotation of the subcellular localization of the protein targeted by the HPA antibody. The following probes/organelles are used as references; (i) DNA stain DAPI for the nucleus, (ii) tubulin as internal control for fixation quality and homogeneity, and (iii) calreticulin for the endoplasmatic reticulum (ER).
The resulting confocal images are single slice images representing one optical section of the cells. The microscope settings are adjusted for each sample to always use the full dynamic range of the detector. The different organelle probes are displayed as different colors/channels in the multicolor images; the HPA antibody staining is shown in green, nuclear stain in blue, micro-tubules in red and ER in yellow.
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Western blot analysis of antibody specificity has been done using a routine sample setup composed of IgG/HSA-depleted human plasma and protein lysates from a limited number of human tissues and cell lines. Antibody binding was visualized by chemiluminescence detection in a CCD-camera system using a peroxidase (HRP) labelled secondary antibody.
Antibodies included in the Protein Atlas have been analyzed using a standardized protocol in a single attempt without further efforts to optimize the procedure and therefore it cannot be excluded that certain observed binding properties are due to technical rather than biological reasons and that further optimization could result in a different outcome.
Version: 3.1 Atlas updated: 2008-02-15 (release history)
Atlas content: 3014 antibodies and 2,940,744 images.
home | about protein atlas - description
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