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, including Triple A Polyclonals and PrecisA Monoclonals, are validated through Immunocytochemistry (ICC-IF) staining in human cell lines, followed by confocal microscopy. This method enables precise determination of the subcellular distribution of the target proteins.
Selection of Cell Lines
To ensure that the protein is endogenously expressed in the target cell lines (eliminating false positives caused by overexpression), we select cell lines with proven endogenous expression of the target protein. We base this selection on RNA-seq data from the Subcellular Section of the Human Protein Atlas, which provides genome-wide RNA expression profiles for 1,206 human cell lines, including 1,132 cancer cell lines.
ICC-IF Staining Process
The selected cell line is cultured in vitro, then fixed and permeabilized using formaldehyde and detergent treatments. The cells are immunofluorescently stained following standardized protocols. In addition to the target antibody, cells are stained for microtubules and counterstained with DAPI to visualize the nucleus.
A high-resolution, three-color image is acquired for each antibody using a Leica SP8X confocal laser scanning microscope with a 63x magnification objective (water or oil immersion). The resulting images represent a single optical section of the analyzed cells, with the following color coding:
Green: antibody staining
Blue: nuclear staining (DAPI)
Red: microtubules
These images are displayed on the product page for each antibody validated in ICC-IF.
Annotation of Subcellular Location
The subcellular localization patterns observed in ICC-IF provide detailed insights into the protein's distribution within the cell, enhancing the characterization and specificity analysis of our antibodies. These patterns are compared with IHC staining data and other experimental protein characterization information to further validate the antibody's specificity and functionality.