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Mice were transcardially perfused with phosphate-buffered saline followed by 4% paraformaldehyde in phosphate buffer. Thirty µm serial sections were cut in a freezing microtome and transferred to phosphate-buffered saline. Free-floating sections were incubated with 1% hydrogen peroxide to quench endogenous peroxidase activity, blocked in 5% normal goat serum, then stained with a rabbit polyclonal antibody to calbindin D-28K (Chemicon) at a 1:1000 dilution. After washing, sections were incubated with Alexa Fluor® 488 goat anti-rabbit IgG antibody (Prod # A11008) at 5 µg/mL (upper panel) or HRP-goat anti-rabbit IgG antibody at 1 µg/mL, followed by Alexa Fluor® 488 tyramide (in TSA Kit #12, T20922; lower panel). Sections were washed, mounted on slides, coverslipped with ProLong® antifade reagent (in Kit P7481) and imaged under identical conditions (10X magnification, 250 millisecond exposure) using a bandpass filter set appropriate for fluorescein (FITC).
|Tested species reactivity||Rabbit|
|Host / Isotype||Goat / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 488|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Cross Adsorption||Against human IgG, human serum, mouse IgG, mouse serum and bovine serum|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1-10 µg/mL|
|Immunocytochemistry (ICC)||1-10 µg/mL|
|Immunofluorescence (IF)||1-10 µg/mL|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Alexa Fluor dyes are among the most trusted fluorescent dyes available today. Invitrogen™ Alexa Fluor™ 488 dye is a bright, green-fluorescent dye with excitation ideally suited to the 488 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 488 dye is pH-insensitive over a wide molar range. Probes with high fluorescence quantum yield and high photostability allow detection of low-abundance biological structures with great sensitivity. Alexa Fluor 488 dye molecules can be attached to proteins at high molar ratios without significant self-quenching, enabling brighter conjugates and more sensitive detection. The degree of labeling for each conjugate is typically 2-8 fluorophore molecules per IgG molecule; the exact degree of labeling is indicated on the certificate of analysis for each product lot.
The goat anti-rabbit IgG whole antibody conjugates are most commonly prepared by immunizing the host animal with a pooled population of immunoglobulins from the target species and can be further purified and modified (e.g., immunoaffinity chromatography, antibody fragmentation, label conjugation, etc.) to generate highly specific reagents. In the first round of purification, whole immunoglobulins binding to the immunizing antibody are recovered and mainly consist of the ~150-kDa IgG class. Further purification with Protein A or G removes all immunoglobulin classes except IgG such that the affinity-purified antibodies react with IgG heavy chains and all classes of immunoglobulin light chains from rabbit. To minimize cross-reactivity, these goat anti-rabbit whole antibodies have been cross-adsorbed against human IgG, human serum, mouse IgG, mouse serum, and bovine serum. Cross-adsorption or pre-adsorption is a purification step to increase specificity of the antibody resulting in higher sensitivity and less background staining. The secondary antibody solution is passed through a column matrix containing immobilized serum proteins from potentially cross-reactive species. Only the nonspecific-binding secondary antibodies are captured in the column, and the highly specific secondaries flow through. The benefits of this extra step are apparent in multiplexing/multicolor-staining experiments where there is potential cross-reactivity with other primary antibodies or in immunohistochemistry experiments where there are may be the presence of endogenous immunoglobulins. For a highly cross-adsorbed secondary antibody equivalent (or equivalent secondary antibody preparation), please see product catalog number: A11034.
Using conjugate solutions: Centrifuge the protein conjugate solution briefly in a microcentrifuge before use; add only the supernatant to the experiment. This step will eliminate any protein aggregates that may have formed during storage, thereby reducing nonspecific background staining. Because staining protocols vary with application, the appropriate dilution of antibody should be determined empirically. For the fluorophore-labeled antibodies a final concentration of 1-10 µg/mL should be satisfactory for most immunohistochemistry and flow cytometry applications.
We offer an extensive line of Invitrogen™ secondary antibody conjugates with well-characterized specificity and labeled with a wide selection of premium fluorescent dyes, including Invitrogen™ Alexa Fluor™ fluorescent dyes. Fluorescent secondary antibody conjugates are useful in the detection, sorting, or purification of its specified target and ideal for fluorescence microscopy and confocal laser scanning microscopy, flow cytometry, and fluorescent western detection. The breadth of fluorescent markers we offer allows our reagents to be tailored to almost any fluorescent detection system.
Secondary antibodies may be provided in three formats: whole IgG, divalent F(ab')2 fragments, and monovalent Fab fragments. Because of the high degree of conservation in the structure of many immunoglobulin domains, most class-specific secondary antibodies must be affinity-purified and cross-adsorbed to achieve minimal cross-reaction with other immunoglobulins.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.