Immunofluorescence cytochemistry (ICC-IF)
How our antibodies are validated in ICC-IF, plus the protocol you need.
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.
4 steps: Cell line → Staining → Imaging → Annotation
| Step | What we do | Why |
|---|---|---|
| 1. Cell line | Choose a human cell line that naturally expresses the target, based on Human Protein Atlas RNA-seq data from 1,206 cell lines | No overexpression, so no false positives |
| 2. Staining | Fix with formaldehyde, permeabilize with detergent, then stain with three markers | Standardized for every antibody |
| 3. Imaging | Leica SP8X confocal microscope, 63x objective, single optical section | High-resolution view inside the cell |
| 4. Annotation | Our experts annotate the subcellular location and compare it with IHC and other protein data | Confirms specificity |
Reading the images
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: our antibody
🔴 Red: microtubules
🔵 Blue: nucleus (DAPI)
You'll find images on the product page of every ICC-IF-validated antibody.
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.
ICC-IF Protocol
Optimized for Triple A Polyclonals and PrecisA Monoclonals