In order to provide an interpretation of the staining patterns, all images generated by ICC-IF are manually annotated. The different organelle probes are displayed as different channels in the multicolor images the HPA antibody staining is shown in green, nucleus in blue, microtubules in red and ER in yellow. The microscope settings are standardized, but the detector gain is optimized for each sample. The resulting confocal images are single slice images representing one optical section of the cells. 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 each gene, the use of PFA or methanol, as well as dilution factors for the antibodies, are stated in the Antibodies and Validation section.
For the great majority of antibodies, fixation is achieved with paraformaldehyde (PFA), but for a few antibodies, this is replaced by methanol (3 times 5 minutes) in order to better preserve the morphology of certain cellular structures. The standard immunostaining protocol for ICC can be found on the open access repository for science methods at protocols.io. In addition to the human cell lines, many proteins have been stained in the mouse cell line NIH 3T3, given that the human and mouse genes are orthologous. Two additional cell lines are selected based on mRNA expression data. In order to localize the whole human proteome on a subcellular level in one specific cell line, most proteins are stained in U-2 OS. Information regarding sex and age of the donor, cellular origin and source is listed here. The selection was furthermore based on morphological characteristics and widespread use of these cell lines. tumor cell lines from mesenchymal, epithelial and glial tumors, as well as cell lines that have immortalized by introduction of telomerase.
The cell line panel has since been expanded to cover more cell types and lineages, e.g. Originally three cell lines, U-2 OS, 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. This provides spatial information on protein expression patterns to define the subcellular localization to cellular organelles and structures at single cell level. The Subcellular Section displays high-resolution, multicolor images of proteins labeled by indirect immunocytochemistry/immunofluorescence (ICC-IF).