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Immunofluorescent analysis of Pax3 (red) in human neural stem cells derived from PD-3 iPSCs using Gibco® PSC Neural Induction Medium (Product # A1647801). The cells were fixed and permeabilized using Image-IT® Fixation/Permeabilization kit (Product # R37602), and blocked with blocking buffer included the kit for one hour at room temperature. Cells were stained with a Pax3 polyclonal antibody (Product # PA1-107) at a dilution of 1:200 in blocking buffer for 3 hours at room temperature, and then incubated with an AlexaFluor 594- conjugated donkey anti-rabbit IgG secondary antibody (Product # A21207) at a dilution of 1:250 for 1 hour at room temperature. Nuclei (blue) were stained with DAPI (Product # D1306). Images were taken on an EVOS® FLoid® Cell Imaging Station at 10X magnification.
|Tested species reactivity||Rabbit|
|Published species reactivity||Not Applicable|
|Host / Isotype||Donkey / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 594|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1-10 µg/mL|
|Immunocytochemistry (ICC)||1-10 µg/ml|
|Immunohistochemistry (IHC)||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.
To minimize cross-reactivity, these donkey anti-rabbit IgG whole antibodies have been affinity-purified and show minimum cross-reactivity to bovine, chicken, goat, guinea pig, hamster, horse, human, mouse, rat, and sheep serum proteins. 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 (e.g., flow cytometry) where there is potential cross-reactivity with other primary antibodies or in tissue/cell fluorescent staining experiments where there may be the presence of endogenous immunoglobulins.
Alexa Fluor dyes are among the most trusted fluorescent dyes available today. Invitrogen™ Alexa Fluor 594 dye is a bright, red-fluorescent dye with excitation ideally suited to the 594 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 594 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 594 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.
Using conjugate solutions: Centrifuge the protein conjugate solution briefly in a microcentrifuge before use; add only the supernatant to the experiment. This step will help 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.
Our secondary 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, for example, with Protein A or G, removes all unwanted immunoglobulin classes except the affinity-purified antibodies that react with the target-specific immunoglobulin heavy and/or light chains.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Hepatocellular carcinoma originates from hepatocytes and not from the progenitor/biliary compartment.
A-21207 was used in immunohistochemistry - paraffin section to elucidate the origin of hepatocellular carcinoma.
|Mu X,Español-Suñer R,Mederacke I,Affò S,Manco R,Sempoux C,Lemaigre FP,Adili A,Yuan D,Weber A,Unger K,Heikenwälder M,Leclercq IA,Schwabe RF||The Journal of clinical investigation (125:3891)||2015|
Autophagy supports survival and phototransduction protein levels in rod photoreceptors.
A-21207 was used in immunohistochemistry - frozen section to investigate the impact of autophagy on photoreceptors.
|Zhou Z,Doggett TA,Sene A,Apte RS,Ferguson TA||Cell death and differentiation (22:488)||2015|
Expression of peroxisome proliferator-activated receptor-gamma in the substantia nigra of hemiparkinsonian nonhuman primates.
A-21207 was used in immunohistochemistry - frozen section to assess PPAR-gamma as a target for neuroprotection in hemiparkinsonian monkeys.
|Swanson C,Emborg M||Neurological research (36:634)||2014|
Piezo2 expression in corneal afferent neurons.
A-21207 was used in immunohistochemistry to investigate the role of Piezo in guinea pig sensory neurons.
|Bron R,Wood RJ,Brock JA,Ivanusic JJ||The Journal of comparative neurology (522:2967)||2014|
|Not Applicable||Not Cited||EdU, a new thymidine analogue for labelling proliferating cells in the nervous system.||Chehrehasa F,Meedeniya AC,Dwyer P,Abrahamsen G,Mackay-Sim A||Journal of neuroscience methods (177:122)||2009|
|Not Applicable||Not Cited||Helicobacter pylori evolution during progression from chronic atrophic gastritis to gastric cancer and its impact on gastric stem cells.||Giannakis M,Chen SL,Karam SM,Engstrand L,Gordon JI||Proceedings of the National Academy of Sciences of the United States of America (105:4358)||2008|
|Not Applicable||Not Cited||Neutralization of human papillomavirus with monoclonal antibodies reveals different mechanisms of inhibition.||Day PM,Thompson CD,Buck CB,Pang YY,Lowy DR,Schiller JT||Journal of virology (81:8784)||2007|
|Not Applicable||Not Cited||NKT cells are critical to initiate an inflammatory response after Pseudomonas aeruginosa ocular infection in susceptible mice.||Hazlett LD,Li Q,Liu J,McClellan S,Du W,Barrett RP||Journal of immunology (Baltimore, Md. : 1950) (179:1138)||2007|
|Not Applicable||Not Cited||Virus-specific CD8+ T cells accumulate near sensory nerve endings in genital skin during subclinical HSV-2 reactivation.||Zhu J,Koelle DM,Cao J,Vazquez J,Huang ML,Hladik F,Wald A,Corey L||The Journal of experimental medicine (204:595)||2007|
|Not Applicable||Not Cited||Coactivation of the N-terminal transactivation of mineralocorticoid receptor by Ubc9.||Yokota K,Shibata H,Kurihara I,Kobayashi S,Suda N,Murai-Takeda A,Saito I,Kitagawa H,Kato S,Saruta T,Itoh H||The Journal of biological chemistry (282:1998)||2007|
|Not Applicable||Not Cited||Characterization of leptin-responsive neurons in the caudal brainstem.||Ellacott KL,Halatchev IG,Cone RD||Endocrinology (147:3190)||2006|
|Not Applicable||Not Cited||Layer acquisition by cortical GABAergic interneurons is independent of Reelin signaling.||Pla R,Borrell V,Flames N,Marín O||The Journal of neuroscience : the official journal of the Society for Neuroscience (26:6924)||2006|
|Not Applicable||Not Cited||Rotavirus NSP4 induces a novel vesicular compartment regulated by calcium and associated with viroplasms.||Berkova Z,Crawford SE,Trugnan G,Yoshimori T,Morris AP,Estes MK||Journal of virology (80:6061)||2006|
|Not Applicable||Not Cited||Granzyme A induces caspase-independent mitochondrial damage, a required first step for apoptosis.||Martinvalet D,Zhu P,Lieberman J||Immunity (22:355)||2005|
|Not Applicable||Not Cited||A rapid enzymatic method for the isolation of defined kidney tubule fragments from mouse.||Wagner CA,Lükewille U,Valles P,Breton S,Brown D,Giebisch GH,Geibel JP||Pflu¿gers Archiv : European journal of physiology (446:623)||2003|
|Not Applicable||Not Cited||A new blocking method for application of murine monoclonal antibody to mouse tissue sections.||Lu QL,Partridge TA||The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society (46:977)||1998|