Antibody Validation for Immunofluorescence
Immunofluorescence (IF) involves the labeling of cellular proteins with specific primary antibodies and fluorochrome-conjugated secondary antibodies (indirect method) or labeling with directly conjugated primary antibodies (direct method). Most fluorescence microscopes allow the examination of subcellular localization, relative expression level and/or activation-state (e.g. phosphorylation status) of two or three fluorescently-labeled proteins in a single sample. The ability to perform multiplexed analyses eliminates the need for consecutive sections when studying a subset of cells with multiple markers in a tissue, saving both time and reagents. Scientists at Cell Signaling Technology (CST) have validated over 800 activation-state specific (e.g., phosphorylation-specific) and total protein antibodies for IF applications, such as manual fluorescence microscopy or automated imaging and laser scanning high content platforms. All CST™ antibodies that are approved for use in immunofluorescent assays have undergone a rigorous validation process.
Validation Steps Include
- Cell lines or tissues with known target expression levels are used to verify specificity.
- Appropriate cell lines and tissues are used to verify subcellular localization.
- Antibody performance is assessed on appropriate tissues.
- Cells are subjected to phosphatase treatment to verify phospho-specificity. Target specificity is also verified with the use of known knockout or null cell lines.
- Cells are subjected to siRNA treatment or over-expression of the target protein to verify target specificity.
- Activation state specification, target expression, and translocation are examined using ligands or inhibitors to modulate pathway activity.
- Requirement of threshold signal-to-noise ratio in antibody:isotype comparison and minimum fold-induction for phospho-specific antibodies ensures the greatest possible sensitivity.
- Fixation and permeabilization conditions are optimized; alternative protocols are recommended if necessary.
- Stringent testing ensures lot-to-lot consistency.