Immunocytochemistry
Learn how our antibodies are validated in immunocytochemistry, and find the protocols you need to be successful in your experiment.
Protocols and guides
In order to achieve the best results with our antibodies, make sure to use these recommended protocols. The protocols are optimized for Triple A Polyclonals and PrecisA Monoclonals.
Validation and characterization in ICC
Our antibodies, Triple A Polyclonals and PrecisA Monoclonals, are characterized and validated by Immunofluorescence (ICC-IF) staining in human cell lines by confocal microscopy, making it possible to pinpoint the subcellular distribution of the analyzed target proteins.
Selection of cell lines
By making use of RNA-seq data for the target genes, which is published for a large panel of cell lines on the Cell Atlas section of the Human Protein Atlas, a cell line with endogenous expression of the target gene is selected for ICC-IF testing. This way, we ensure that the protein target is endogenously expressed in the target cell lines, thereby eliminating any false positive staining that could be caused by the artificial overexpression of target proteins.
ICC staining
The selected cell line is cultured in vitro, fixed/permeabilized using formaldehyde/detergent treatment and immunofluorescently stained according to standardized protocols.
In addition to the target-specific antibody to be analyzed, cells are also stained with organelle markers specific for microtubules as well as counterstained with the nuclear probe DAPI. A high-resolution, three-color image, is acquired for each antibody using a Leica SP8X confocal laser scanning microscope equipped with a 63x magnification, water or oil immersion objective.
The resulting images are single slice images representing one optical section of the analyzed cells. The different organelle probes are displayed as different channels in the multicolor images; the target antibody staining is shown in green, nuclear stain in blue and microtubules in red. The images are presented on the product data page for each ICC validated antibody.
Annotation of subcellular location
The detailed information of the subcellular localization for a protein improves the characterization and specificity analysis of the antibodies beyond what is possible with immunohistochemistry (IHC). The subcellular localization patterns are compared with the IHC staining as well as the experimental protein characterization data available.
Triple A Polyclonals on the Cell Atlas
Our Triple A Polyclonals were used for the creation of the Cell Atlas section of the Human Protein Atlas, where the antibodies were used to determine the subcellular distribution of a large percentage of the human proteome.
Antibodies were used in ICC-IF staining of three human cell lines and imaged by confocal microscope, making it possible to pinpoint the subcellular distribution of the analyzed target proteins. For all antibodies that were to be analyzed, two positive control cell lines were selected based on RNA sequencing data in addition to U-2 OS that was included for all analyzed antibodies.
The extensive characterization data generated in the Human Protein Atlas project is available in the open-access Cell Atlas. On the product page for each Triple A Polyclonal validated for ICC, you can find links to this data on the Human Protein Atlas.
Explore the Cell Atlas
Our Triple A Polyclonals were used for the creation of the Cell Atlas section of the Human Protein Atlas, where the antibodies were used to determine the subcellular distribution of a large percentage of the human proteome.