Immunofluorescence Validation
Confocal immunofluorescent analysis of HT-1080 cells using Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (D13A11) Rabbit mAb (green), β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 555 Conjugate) #2116 (red), and Phospho-Histone H3 (Ser10) (6G3) Mouse mAb #9706 (blue).
Confocal IF analysis of 3T3-L1 adipocytes, isoproterenol-treated (left) or phosphatase-treated (right), using Phospho-HSL (Ser563) Antibody #4139 (red). Lipid droplets have been labeled with BODIPY® 493/503 (green). Blue = DRAQ5™ fluorescent DNA dye.
Activation State-specific Antibodies Validated for Immunofluorescence
Immunofluorescence (IF) involves the labeling of cellular proteins in cells 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.
Our IF protocol involves formaldehyde fixation to stabilize epitopes and subsequent immunostaining in a buffer containing Triton X-100. This detergent permeabilizes the cells resulting in enhanced antibody penetration and decreased autofluorescence. Alternatively, some antibodies (e.g. phospho-Stats) require methanol-permeabilization for proper staining.
At CST, all IF-validated antibodies have undergone a rigorous validation protocol that utilizes titration to determine optimal concentration and screening of known positive and negative control cells/tissues to verify specificity. Labeled slides are examined using high-resolution confocal microscopy to confirm localization and assess areas of nonspecific staining. Whenever possible, antibodies are validated on various cell/tissue preparations – cells grown on chamber slides, coverslips or in image-quality multi-well plates (IF-IC), floating or slide-mounted frozen sections (IF-F), or formaldehyde-fixed paraffin-embedded tissue samples (IF-P). Many IF-validated antibodies can also be used for *In-Cell Western™, high throughput or high content screening.
Our goal is to provide customers with images clearly demonstrating correct localization and outstanding performance for all IF-validated antibodies, optimized fixation and staining protocols and helpful technical support from knowledgeable experienced scientists. If you would like assistance selecting an antibody for an application or have questions about protocols, you can contact our IF scientists directly at info@cstj.co.jp.
*Data may be generated with methods/devices/compositions requiring a license to relevant third party intellectual property rights.