Immunocytochemistry/IF - cells

The subcellular resource revolves around high-resolution, multicolor images of proteins labeled by indirect immunocytochemistry/immunofluorescence (ICC-IF). This provides spatial information on protein localization in terms of the subcellular distribution of the protein in organelles and subcellular structures at single cell level.

Data generation

Three cell lines, originally U2OS, A-431 and U-251 MG, originating from different human tissues were chosen to be included in the analysis of protein subcellular localization by ICC-IF. The cell line panel has since been expanded to cover more cell types and lineages, e.g. tumor cell lines from mesenchymal, epithelial and glial tumors, as well as cell lines that have immortalized by introduction of telomerase. The selection was furthermore based on morphological characteristics and widespread use of these cell lines. Information regarding sex and age of the donor, cellular origin and source is listed here. In order to localize the whole human proteome on a subcellular level in one specific cell line, most proteins are stained in U2OS. Two additional cell lines are selected based on mRNA expression data. Some proteins have also been stained in one or more ciliated cells lines and/or in human sperm, originating from a single healthy donor. In addition to the human cells, many proteins have been stained in the mouse cell line NIH 3T3, given that the human and mouse genes are orthologous.

The standard immunostaining protocol for ICC can be found on the open access repository for science methods at protocols.io. For the great majority of antibodies, fixation is achieved with paraformaldehyde (PFA), but for a few antibodies, this is replaced by methanol in order to better preserve the morphology of certain cellular structures. For each gene, the use of PFA or methanol, as well as dilution factors for the antibodies, are stated in the Antibodies and Validation section. In order to facilitate the annotation of the subcellular localization of the protein targeted by the HPA antibody, the cells are also stained with reference markers: (i) DAPI for the nucleus, (ii) anti-tubulin antibody for microtubules, and (iii) anti-calreticulin or anti-KDEL for the endoplasmic reticulum (ER). For ciliated cells lines, an antibody targeting ARL13B has been used to mark primary cilia and an antibody targeting pericentrin (PCNT) has been used to mark basal bodies. In human sperm, an antibody targeting acetylated tubulin has been used as a marker for flagella and an antibody targeting citrate synthase (CS) has been used as a marker for mitochondria.

The resulting confocal images are single slice images representing one optical section of the cells, except for ciliated cell lines and human sperm, in which case z-stacks are shown. The microscope settings are standardized, but the detector gain is optimized for each sample. The different organelle probes are displayed as different channels in the multicolor images, with the HPA antibody staining shown in green, nucleus in blue, microtubules in red and ER in yellow.

Annotation

In order to provide an interpretation of the staining patterns, all images generated by ICC-IF are manually annotated. For each cell line and antibody, the staining is described in terms of subcellular location(s) and single-cell variability (SCV). The table below lists the subcellular locations used for annotation, with links to the cell structure dictionary entry and corresponding GO terms. SCVs within an immunofluorescence image are classified as intensity variation (variation in their expression level) or as spatial variation (variation in the spatial distribution).

Knowledge-based annotation

The knowledge-based annotation aims to provide an interpretation of the detected subcellular localization of a protein. In the first step, stainings in different cell lines with the same antibody are reviewed and the results are compared with external experimental protein/gene characterization data for subcellular localization, available in the UniProtKB/Swiss-Prot database. In the second step, all antibodies targeting the same protein are taken in consideration for a final annotation of the subcellular distribution of the protein.

Reliability score

Each location is separately given one of the four reliability scores (Enhanced, Supported, Approved, or Uncertain) based on available protein/RNA/gene characterization data from both HPA and the UniProtKB/Swiss-Prot database. The reliability score also encompass several additional factors, including reproducibility of the antibody staining in different cell lines, correlation between staining intensity and RNA expression levels, and assays for enhanced antibody validation. Enhanced validation is achieved by using antibodies binding to different epitopes on the same target protein (independent antibody validation), by assessing staining intensity upon knockdown/knockout of the target protein (genetic validation) and/or by matching of the signal with a GFP-tagged protein (recombinant expression validation), and experimental evidence for subcellular location described in literature. The individual location relibility scores are summarized in an overall gene reliability score.

There are four different reliability scores:

Enhanced - The antibody has enhanced validation and there is no contradicting data, such as literature describing experimental evidence for a different location.
Supported - There is no enhanced validation of the antibody, but the annotated localization is reported in literature.
Approved - The localization of the protein has not been previously described and was detected by only one antibody without additional antibody validation.
Uncertain - The antibody-staining pattern contradicts experimental data or expression is not detected at RNA level.