Immunofluorescence cytochemistry (ICC-IF)

Learn how our antibodies are validated in immunocytochemistry, and find the protocols you need to be successful in your experiment.

Protocols and guides

Use these recommended protocols for optimal results in ICC-IF using our antibodies. The protocols are optimized for Triple A Polyclonals and PrecisA Monoclonals.

ICC-IF Standard Protocol

Validation and characterization in ICC-IF

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

How do we ensure that the protein is endogenously expressed in the target cell lines, thereby eliminating any false positive staining caused by the artificial overexpression of proteins?

For ICC-IF testing we select the cell line(s) with a proven endogenous expression of the target gene, using the RNA-seq data published on the Subcellular Section of the Human Protein Atlas.

The Cell Line section contains information on genome-wide RNA expression profiles of human protein-coding genes in 1206 human cell lines, including 1132 cancer cell lines.

The Cell Line Database

ICC-IF 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 specific antibody, cells are stained for microtubules and 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 antibody staining is shown in green,
• the nuclear stain is shown as blue,
• the microtubules are shown in red.
  The images are displayed on the product page for each antibody validated in ICC.

Annotation of subcellular location

The detailed information on the subcellular localization of 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 to map all the human proteins within the Human Protein Atlas project, including the Subcellular Atlas and Cell Line Atlas.

For all antibodies validated in ICC, we selected three human cell lines (two positive control and one U-2 OS cell line) based on RNA sequencing data for the specific antibodies.

The extensive characterization data generated in the Human Protein Atlas project is freely available. You can find links to the images on Human Protein Atlas on each product page.

 

Explore Triple A Polyclonals

 

All Protocols and guides

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. See all our protocols for IHC, WB, and ICC-IF.

Go to protocols