Antibody Validation for Immunofluorescence-based Assays
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.
Phosphatase Treatment and Knockout Cells
Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb #5558: Confocal immunofluorescent analysis of wild type mouse embryonic fibroblasts (MEFs) (upper row), GSK-3β (-/-) MEFs (middle row), or PC-3 cells (lower row), untreated (left), treated with LY294002 (PI3 Kinase Inhibitor) #9901 and Wortmannin #9951 (center) or λ phosphatase-treated (right), using Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb #5558 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). (MEF wild type and GSK-3β (-/-) cells were kindly provided by Dr. Jim Woodgett, University of Toronto, Canada).
Subcellular Localization
Figure A. - COX IV (3E11) Rabbit mAb #4850
Figure B. - ERp72 (D70D12) XP® Rabbit mAb #5033
Figure C. - ZO-3 (D57G7) XP® Rabbit mAb #3704
Tissue
Pdx1 Antibody #2437: Confocal immunofluorescent analysis of normal rat pancreas using Pdx1 Antibody #2437 (green, upper) or Insulin (C27C9) Rabbit mAb #3014 (green, lower). Keratin filaments were labeled with Pan-Keratin (C11) Mouse mAb (Alexa Fluor® 647 Conjugate) #4528 (blue). Red = Propidium Iodide/RNase #4087 (fluorescent DNA dye).
Protocol Optimization
PDI Antibody #2446 and β-Actin (8H10D10) Mouse mAb #3700: Confocal IF analysis of NIH/3T3 cells, permeabilized with methanol (upper) or 0.3% Triton X-100 (lower), using #2446 (green) and #3700 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
