|Product Name||Goat Anti-Rabbit IgG（H+L） Secondary Antibody, Cy3 Conjugate|
|Synonyms||Cy3-conjugated Goat Anti-Rabbit IgG; Goat Anti-Rabbit IgG-Cyanine 3 Secondary Antibody; Cy3-labeled Goat Anti-Rabbit IgG Secondary Antibody|
|Description||Goat Anti-Rabbit IgG Secondary Antibody, Cy3 Conjugate, for detection, localization and quantification of target proteins in sliced samples via indirect immunofluorescence in IHC-P, IHC-F or ICC.|
|Reagent Type||Fluorophore-conjugated secondary antibody|
|Immunogen||Whole molecule rabbit IgG|
|Specificity||Rabbit IgG specific|
|Form Supplied||Liquid: concentrated buffered stock solution|
|Formulation||0.5 mg Cy3-conjugated secondary antibody |
0.01 M PBS (PH 7.4)
|Application||Immunohistochemistry (IHC-P and IHC-F), Immunocytochemistry (ICC) |
|Storage||4C for 1 year|
|Precautions||FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR CLINICAL USE|
|Sample Type||Mouse primary-antibody-probed formalin-fixed paraffin-embedded (FFPE) tissue sections on slides (IHC-P), or thawed frozen samples (IHC-F)|
|Assay Purpose||Protein detection/quantification|
|Equipment Needed||Excitation light source, filter set and detector, fluorescence microscope (can be combined with confocal microscope), fluorescence plate-reader|
|Specific||High signal-to-noise ratio|
|High Signal Amplification||Multiple secondary antibodies can bind to a single primary antibody, multiple Cy3 molecules bind to a single secondary antibody|
|Fast||Fewer processing steps - no need to add a substrate; Less optimization required compared to enzymatic detection; Generates strong signals in a relatively short time span; signal can be observed directly|
|Absolute Quantification||Precise quantitative analysis of fluorescence microscopy images can provide absolute protein amount and information regarding stoichiometry of protein complexes|
|Stability||-Cyanine dyes are better able to withstand the harsh dehydration and embedding conditions required for mounting sections |
-Reduced fluorescence quenching (sulfonated cyanines are less prone to aggregation in water due to reduced dye-dye interactions)
-Cy3 conjugates are bright orange-red and more photostable in the aqueous environment, as well as in non-polar media
|Superb Signal Detection||Precise target localization, high extinction coefficient, and good quantum yield of Cy3 results in brighter fluorescence and less background than other orange-red fluorescing dye conjugates|
|Flexible||No need to label each antibody against each target protein with a fluorescent dye|
|Multiplex Compatible||Compatible with colocalization studies (multiple antigens concurrent detection) even in close proximity using primary antibodies from different host species for simultaneous detection by fluorophore-conjugated secondary antibodies, or using multiple differently colored fluorophores (FITC and TRITC) in the same experiment for target differentiation.|
Most commonly, secondary antibodies are generated by immunizing the host animal with a pooled population of immunoglobulins from the target species. The host antiserum is then purified through immunoaffinity chromatography to remove all host serum proteins, except the specific antibody of interest, and then modified with antibody fragmentation, label conjugation, etc., to generate highly specific detection reagents. Secondary antibodies can be conjugated to a large number of labels, including enzymes, biotin, fluorescent dyes/proteins, or gold particles. Here, the antibody provides the specificity to locate the protein of interest by recognizing a primary antibody that targets a particular antigen, and the label generates a detectable signal in the area of the formed immune complexes. The label of choice depends upon the experimental application.
Fluorescent reporters widely used in biological research are of two types: organic compounds with a low molecular weight (0.2-1 kDa) typically containing numerous aromatic groups, or plane or cyclic moieties with π bonds (e.g.FITC, TRITC, Alexa Fluor Dyes, DyLight Fluors), and biological fluorophores (e.g.green fluorescent protein (GFP), R-Phycoerythrin). Cy3 is a cyanine dye with 3 carbon atoms/methine groups between two indolenine groups, NHS-ester, sulfonated at both indoles. It fluoresces greenish yellow with excitation max at ~550 nm and emission at ~570 nm. Cy3 can be detected by various fluorometers, imagers, and microscopes with standard filters for Tetramethylrhodamine (TRITC). Due to inherently high extinction coefficient, this dye is also easily detected by naked eye on gels, and in solution. Cy3 can be excited to about 50% of maximum with an argon laser (514 nm or 528 nm lines), or to about 75% of maximum with a helium/neon laser (543 nm line) or mercury lamp (546 nm line). For double labeling Cy3 has been typically used with Cy5, with fluorescein (using narrow band-pass emission filter to minimize Cy3 fluorescence in the FITC filter set), and also with with Alexa Fluor 647 for multiple labeling when using a confocal microscope. Analog of Cy3 with greater photostability and higher fluorescence intensity suited for various biological applications such as imaging and flow cytometry, is Alexa Fluor 555 dye.
MCM2 was detected in paraffin-embedded sections of human intestinal cancer tissues using rabbit anti- MCM2 Antigen Affinity purified polyclonal antibody at 1 μg/mL. Cy3 Conjugated Goat Anti-Rabbit IgG secondary antibody (Catalog # BA1032) was used to detect the primary antibody at 20μg/mL.
Collagen IV was detected in paraffin-embedded sections of rat kidney tissues using rabbit anti- Collagen IV Antigen Affinity purified polyclonal antibody at 1 μg/mL. Cy3 Conjugated Goat Anti-Rabbit IgG secondary antibody (Catalog # BA1032) was used to detect the primary antibody at 20 μg/mL.