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Immunofluorescence analysis of Rabbit anti-Mouse IgG (H+L) Cross Adsorbed Secondary Antibody, DyLight 488 was performed using MCF-7 cells stained with Cytokeratin 19 (RCK108) Mouse Monoclonal Primary Antibody (MA512613). 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. Rabbit anti-Mouse IgG (H+L) Cross Adsorbed Secondary Antibody, DyLight 488 (SA510158) was used at a concentration of 1µg/ml in phosphate buffered saline containing 0.2 % BSA for 45 minutes at room temperature, for detection of Cytokeratin 19 in the cytoplasm (Panel a: green). Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (S36938). F-actin was stained with Rhodamine Phalloidin (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|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Mouse IgG-heavy and light chain|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS with 0.2% BSA|
|Contains||0.09% sodium azide|
|Storage Conditions||4° C|
|Cross Adsorption||Against human and rat|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:25 - 1:100|
|Immunocytochemistry (ICC)||1 µg/ml|
|Immunofluorescence (IF)||1 µg/ml|
|Immunohistochemistry (IHC)||1:50 - 1:2,000|
|Immunoprecipitation (IP)||Assay Dependent|
|Western Blot (WB)||1:5,000 - 1:20,000|
* 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.
This antibody is cross-adsorbed and exhibits minimum reactivity to human and rat.
For Western blot applications, 5% non-fat dry milk in PBST or TBST is recommended for blocking and incubation of antibodies. BSA is not recommended.
Thermo Scientific Anti-Mouse secondary antibodies are affinity-purified antibodies with well-characterized specificity for mouse immunoglobulins and are useful in the detection, sorting or purification of its specified target. Secondary antibodies offer increased versatility enabling users to use many detection systems (e.g. HRP, AP, fluorescence). They can also provide greater sensitivity through signal amplification as multiple secondary antibodies can bind to a single primary antibody. Most commonly, secondary antibodies are generated by immunizing the host animal with a pooled population of immunoglobulins from the target species and can be further purified and modified (i.e. immunoaffinity chromatography, antibody fragmentation, label conjugation, etc.) to generate highly specific reagents.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.