Immunofluorescence analysis of Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate (Product # A21202) was performed using MCF-7 cells stained with Cytokeratin 19 Mouse Monoclonal Antibody (Product # MA5-12613). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, blocked with 1% BSA for 1 hour and labeled with mouse primary antibody (1:250 dilution) for 3 hours at room temperature. Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate was used at concentration of 0.2 µg/ml in phosphate buffered saline containing 0.2 % BSA for 45 minutes at room temperature, for detection of cytokeratin 19 in the membrane (Panel a: green). Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (Product # S36938). F-actin was stained with Rhodamine Phalloidin (Product # R415, 1:300) (Panel c: red). Panel d represents the composite image. No nonspecific staining was observed with the secondary antibody alone (panel f), or with an isotype control (panel e). The images were captured at 60X magnification.
|Tested species reactivity||Mouse|
|Published species reactivity||Not Applicable|
|Host / Isotype||Donkey / 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|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:500|
|Immunocytochemistry (ICC)||0.2 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.
|Miscellaneous PubMed (MISC)||See 34 publications below|
|Immunohistochemistry (Paraffin) (IHC (P))||See 1 publications below|
|Immunocytochemistry (ICC)||See 10 publications below|
|Immunohistochemistry (Frozen) (IHC (F))||See 3 publications below|
|Immunohistochemistry (IHC)||See 2 publications below|
To minimize cross-reactivity, these donkey anti-mouse 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 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.
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.
|Not Applicable||Not Cited||
Investigation into the effects of varying frequency of mechanical stimulation in a cycle-by-cycle manner on engineered cardiac construct function.
A-21202 was used in immunohistochemistry (frozen) to develop a bioreactor system that allows for the control of the mechanical stimulation of engineered cardiac tissue on a cycle-by-cycle basis.
|Morgan KY,Black LD||Journal of tissue engineering and regenerative medicine (11:342)||2017|
|Not Applicable||Not Cited||Technical Advance: The rat aorta contains resident mononuclear phagocytes with proliferative capacity and proangiogenic properties.||Zorzi P,Aplin AC,Smith KD,Nicosia RF||Journal of leukocyte biology (88:1051)||2010|
|Not Applicable||Not Cited||High-content phenotypic profiling of drug response signatures across distinct cancer cells.||Caie PD,Walls RE,Ingleston-Orme A,Daya S,Houslay T,Eagle R,Roberts ME,Carragher NO||Molecular cancer therapeutics (9:1913)||2010|
|Not Applicable||Not Cited||Human stem cell delivery for treatment of large segmental bone defects.||Dupont KM,Sharma K,Stevens HY,Boerckel JD,García AJ,Guldberg RE||Proceedings of the National Academy of Sciences of the United States of America (107:3305)||2010|
|Not Applicable||Not Cited||An impaired transendothelial migration potential of chronic lymphocytic leukemia (CLL) cells can be linked to ephrin-A4 expression.||Trinidad EM,Ballesteros M,Zuloaga J,Zapata A,Alonso-Colmenar LM||Blood (114:5081)||2009|
|Not Applicable||Not Cited||T cell antigen receptor signaling and immunological synapse stability require myosin IIA.||Ilani T,Vasiliver-Shamis G,Vardhana S,Bretscher A,Dustin ML||Nature immunology (10:531)||2009|
|Not Applicable||Not Cited||Bryostatin-5 blocks stromal cell-derived factor-1 induced chemotaxis via desensitization and down-regulation of cell surface CXCR4 receptors.||He X,Fang L,Wang J,Yi Y,Zhang S,Xie X||Cancer research (68:8678)||2008|
|Not Applicable||Not Cited||pLG72 modulates intracellular D-serine levels through its interaction with D-amino acid oxidase: effect on schizophrenia susceptibility.||Sacchi S,Bernasconi M,Martineau M,Mothet JP,Ruzzene M,Pilone MS,Pollegioni L,Molla G||The Journal of biological chemistry (283:22244)||2008|
|Not Applicable||Not Cited||Early resolution of acute immune activation and induction of PD-1 in SIV-infected sooty mangabeys distinguishes nonpathogenic from pathogenic infection in rhesus macaques.||Estes JD,Gordon SN,Zeng M,Chahroudi AM,Dunham RM,Staprans SI,Reilly CS,Silvestri G,Haase AT||Journal of immunology (Baltimore, Md. : 1950) (180:6798)||2008|
|Not Applicable||Not Cited||TRPC channels determine human keratinocyte differentiation: new insight into basal cell carcinoma.||Beck B,Lehen'kyi V,Roudbaraki M,Flourakis M,Charveron M,Bordat P,Polakowska R,Prevarskaya N,Skryma R||Cell calcium (43:492)||2008|
|Not Applicable||Not Cited||Anti-biotin antibodies offer superior organelle-specific labelling of mitochondria over avidin or streptavidin.||Coene ED,Shaw MK,Vaux DJ||Methods in molecular biology (Clifton, N.J.) (418:157)||2008|
|Not Applicable||Not Cited||Sulfated glycosphingolipid as mediator of phagocytosis: SM4s enhances apoptotic cell clearance and modulates macrophage activity.||Popovic ZV,Sandhoff R,Sijmonsma TP,Kaden S,Jennemann R,Kiss E,Tone E,Autschbach F,Platt N,Malle E,Gröne HJ||Journal of immunology (Baltimore, Md. : 1950) (179:6770)||2007|
|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||Simultaneous visualization of multiple antigens with tyramide signal amplification using antibodies from the same species.||Tóth ZE,Mezey E||The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society (55:545)||2007|
|Not Applicable||Not Cited||Src-dependent phosphorylation of membrane type I matrix metalloproteinase on cytoplasmic tyrosine 573: role in endothelial and tumor cell migration.||Nyalendo C,Michaud M,Beaulieu E,Roghi C,Murphy G,Gingras D,Béliveau R||The Journal of biological chemistry (282:15690)||2007|
|Not Applicable||Not Cited||Essential role of PDK1 in regulating endothelial cell migration.||Primo L,di Blasio L,Roca C,Droetto S,Piva R,Schaffhausen B,Bussolino F||The Journal of cell biology (176:1035)||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||TrkA receptor activation by nerve growth factor induces shedding of the p75 neurotrophin receptor followed by endosomal gamma-secretase-mediated release of the p75 intracellular domain.||Urra S,Escudero CA,Ramos P,Lisbona F,Allende E,Covarrubias P,Parraguez JI,Zampieri N,Chao MV,Annaert W,Bronfman FC||The Journal of biological chemistry (282:7606)||2007|
|Not Applicable||Not Cited||DeltaF508 mutation results in impaired gastric acid secretion.||Sidani SM,Kirchhoff P,Socrates T,Stelter L,Ferreira E,Caputo C,Roberts KE,Bell RL,Egan ME,Geibel JP||The Journal of biological chemistry (282:6068)||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||Akt1 deficiency affects neuronal morphology and predisposes to abnormalities in prefrontal cortex functioning.||Lai WS,Xu B,Westphal KG,Paterlini M,Olivier B,Pavlidis P,Karayiorgou M,Gogos JA||Proceedings of the National Academy of Sciences of the United States of America (103:16906)||2006|
|Not Applicable||Not Cited||Evidence for stroke-induced neurogenesis in the human brain.||Jin K,Wang X,Xie L,Mao XO,Zhu W,Wang Y,Shen J,Mao Y,Banwait S,Greenberg DA||Proceedings of the National Academy of Sciences of the United States of America (103:13198)||2006|
|Not Applicable||Not Cited||Matrix metalloproteinase-9 degrades amyloid-beta fibrils in vitro and compact plaques in situ.||Yan P,Hu X,Song H,Yin K,Bateman RJ,Cirrito JR,Xiao Q,Hsu FF,Turk JW,Xu J,Hsu CY,Holtzman DM,Lee JM||The Journal of biological chemistry (281:24566)||2006|
|Not Applicable||Not Cited||Heterogeneous nuclear ribonucleoprotein (hnRNP) E1 binds to hnRNP A2 and inhibits translation of A2 response element mRNAs.||Kosturko LD,Maggipinto MJ,Korza G,Lee JW,Carson JH,Barbarese E||Molecular biology of the cell (17:3521)||2006|
|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||The adaptor protein Nck interacts with Fas ligand: Guiding the death factor to the cytotoxic immunological synapse.||Lettau M,Qian J,Linkermann A,Latreille M,Larose L,Kabelitz D,Janssen O||Proceedings of the National Academy of Sciences of the United States of America (103:5911)||2006|
|Not Applicable||Not Cited||Specific T-type calcium channel isoforms are associated with distinct burst phenotypes in deep cerebellar nuclear neurons.||Molineux ML,McRory JE,McKay BE,Hamid J,Mehaffey WH,Rehak R,Snutch TP,Zamponi GW,Turner RW||Proceedings of the National Academy of Sciences of the United States of America (103:5555)||2006|
|Not Applicable||Not Cited||EphB receptors regulate dendritic spine morphogenesis through the recruitment/phosphorylation of focal adhesion kinase and RhoA activation.||Moeller ML,Shi Y,Reichardt LF,Ethell IM||The Journal of biological chemistry (281:1587)||2006|
|Not Applicable||Not Cited||LPS receptor (CD14): a receptor for phagocytosis of Alzheimer's amyloid peptide.||Liu Y,Walter S,Stagi M,Cherny D,Letiembre M,Schulz-Schaeffer W,Heine H,Penke B,Neumann H,Fassbender K||Brain : a journal of neurology (128:1778)||2005|
|Not Applicable||Not Cited||TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells.||James D,Levine AJ,Besser D,Hemmati-Brivanlou A||Development (Cambridge, England) (132:1273)||2005|
|Not Applicable||Not Cited||Multiplex detection of RNA expression in Drosophila embryos.||Kosman D,Mizutani CM,Lemons D,Cox WG,McGinnis W,Bier E||Science (New York, N.Y.) (305:null)||2004|
|Not Applicable||Not Cited||Biotin is endogenously expressed in select regions of the rat central nervous system.||McKay BE,Molineux ML,Turner RW||The Journal of comparative neurology (473:86)||2004|
|Not Applicable||Not Cited||Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events.||Krutzik PO,Nolan GP||Cytometry. Part A : the journal of the International Society for Analytical Cytology (55:61)||2003|
|Not Applicable||Not Cited||Morphology and dynamics of clathrin/GGA1-coated carriers budding from the trans-Golgi network.||Puertollano R,van der Wel NN,Greene LE,Eisenberg E,Peters PJ,Bonifacino JS||Molecular biology of the cell (14:1545)||2003|
Cancer stem cells in moderately differentiated oral tongue squamous cell carcinoma express components of the renin-angiotensin system.
A-21202 was used in immunohistochemistry - paraffin section to study moderately differentiated oral tongue squamous cell carcinoma that expression of components of the renin-angiotensin system in cancer stem cells
|Itinteang T,Dunne JC,Chibnall AM,Brasch HD,Davis PF,Tan ST||Journal of clinical pathology (69:942)||2016|
|Not Applicable||Not Cited||
Autophagy activation by novel inducers prevents BECN2-mediated drug tolerance to cannabinoids.
A-21202 was used in immunocytochemistry to analyze prevention of BECN2-mediated drug tolerance to cannabioids by autophagy activation by novel inducers
|Kuramoto K,Wang N,Fan Y,Zhang W,Schoenen FJ,Frankowski KJ,Marugan J,Zhou Y,Huang S,He C||Autophagy (12:1460)||2016|
Tau accumulation induces synaptic impairment and memory deficit by calcineurin-mediated inactivation of nuclear CaMKIV/CREB signaling.
A-21202 was used in immunocytochemistry to assess induction of synaptic impairment and memory deficit by calcieurin-mediated inactivation of nuclear CaMKIV/CREB signaling due to tau accumulation
|Yin Y,Gao D,Wang Y,Wang ZH,Wang X,Ye J,Wu D,Fang L,Pi G,Yang Y,Wang XC,Lu C,Ye K,Wang JZ||Proceedings of the National Academy of Sciences of the United States of America (113:E3773)||2016|
Assembly Dynamics and Stoichiometry of the Apoptosis Signal-regulating Kinase (ASK) Signalosome in Response to Electrophile Stress.
A-21202 was used in immunocytochemistry to characterize the response to electrophile stress by assembly stoichiometry and dynamics of the apoptosis signal-regulating kinase (ASK) signalosome
|Federspiel JD,Codreanu SG,Palubinsky AM,Winland AJ,Betanzos CM,McLaughlin B,Liebler DC||Molecular and cellular proteomics : MCP (15:1947)||2016|
Histone chaperone CAF-1 mediates repressive histone modifications to protect preimplantation mouse embryos from endogenous retrotransposons.
A-21202 was used in immunocytochemistry to protect preimplantation mouse embryos from endogenous retrotransposons by histone chaperone CAF-1 mediating repressive histone modifications
|Hatanaka Y,Inoue K,Oikawa M,Kamimura S,Ogonuki N,Kodama EN,Ohkawa Y,Tsukada Y,Ogura A||Proceedings of the National Academy of Sciences of the United States of America (112:14641)||2015|
|Not Applicable||Not Cited||
9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate.
A-21202 was used in immunocytochemistry to analyze catalysis of 9-O-Acetylation of sialic acids by CASD1 through a covalent acetyl-enzyme intermediate
|Baumann AM,Bakkers MJ,Buettner FF,Hartmann M,Grove M,Langereis MA,de Groot RJ,Mühlenhoff M||Nature communications (6:null)||2015|
Basonuclin-1 modulates epithelial plasticity and TGF-ß1-induced loss of epithelial cell integrity.
A-21202 was used in immunocytochemistry to show that Basonuclin- regulates TGF-β-induced epithelial dedifferentiation of mammary epithelial cells.
|Feuerborn A,Mathow D,Srivastava PK,Gretz N,Gröne HJ||Oncogene (34:1185)||2015|
Nucleolar tethering mediates pairing between the IgH and Myc loci.
A-21202 was used in immunocytochemistry to test if NOR-mediated tethering serves as a mechanism to drive IgH:Myc colocalization
|Strongin DE,Groudine M,Politz JC||Nucleus (Austin, Tex.) (5:474)||2014|
The nuclear form of glutathione peroxidase 4 colocalizes and directly interacts with protamines in the nuclear matrix during mouse sperm chromatin assembly.
A-21202 was used in immunocytochemistry to test if nGPx4 directly interacts with protamines by transiently sharing a nuclear matrix localization.
|Puglisi R,Maccari I,Pipolo S,Mangia F,Boitani C||Spermatogenesis (4:null)||2014|
Calreticulin protects rat microvascular endothelial cells against microwave radiation-induced injury by attenuating endoplasmic reticulum stress.
A-21202 was used in immunocytochemistry to examine the effect of CRT treatment on MR-induced injury in rat MMECs.
|Li WH,Li YZ,Song DD,Wang XR,Liu M,Wu XD,Liu XH||Microcirculation (New York, N.Y. : 1994) (21:506)||2014|
|Not Applicable||Not Cited||
Nanopodia--thin, fragile membrane projections with roles in cell movement and intercellular interactions.
A-21202 was used in immunocytochemistry to develop methods to study TM4SF1-derived nanopodia
|Lin CI,Lau CY,Li D,Jaminet SC||Journal of visualized experiments : JoVE (null:null)||2014|
|Not Applicable||Not Cited||
The methyl binding domain 3/nucleosome remodelling and deacetylase complex regulates neural cell fate determination and terminal differentiation in the cerebral cortex.
A-21202 was used in immunohistochemistry - frozen section to investigate the role of MBD3/NuRD in neurogenesis.
|Knock E,Pereira J,Lombard PD,Dimond A,Leaford D,Livesey FJ,Hendrich B||Neural development (10:null)||2015|
Ventral pallidal projections to mediodorsal thalamus and ventral tegmental area play distinct roles in outcome-specific Pavlovian-instrumental transfer.
A-21202 was used in immunohistochemistry - frozen section to study the Pavlovian-instrumental transfer effect of the rostral medial ventral pallidum region innervated by the nucleus accumbens shell
|Leung BK,Balleine BW||The Journal of neuroscience : the official journal of the Society for Neuroscience (35:4953)||2015|
Boundary cap neural crest stem cells homotopically implanted to the injured dorsal root transitional zone give rise to different types of neurons and glia in adult rodents.
A-21202 was used in immunohistochemistry - frozen section to study the fate of mouse boundary cap neural crest stem cells implanted in the dorsal root transitional zone
|Trolle C,Konig N,Abrahamsson N,Vasylovska S,Kozlova EN||BMC neuroscience (15:null)||2014|
Congenital heart disease protein 5 associates with CASZ1 to maintain myocardial tissue integrity.
A-21202 was used in immunohistochemistry to investigate the role of cardiac transcription factor CASTOR (CASZ1) in heart development
|Sojka S,Amin NM,Gibbs D,Christine KS,Charpentier MS,Conlon FL||Development (Cambridge, England) (141:3040)||2014|
Investigation of general and cytoskeletal markers to estimate numbers and proportions of neurons in the human intestine.
A-21202 was used in immunohistochemistry to compare panneuronal markers in the enteric nervous system
|Ganns D,Schrödl F,Neuhuber W,Brehmer A||Histology and histopathology (21:41)||2006|