Goat Anti-Mouse IgG(H+L) Antibody;FITC conjugated

How should Goat Anti-Mouse IgG(H+L)-FITC be stored for optimal performance?

For optimal performance and longevity, Goat Anti-Mouse IgG(H+L)-FITC antibody should be stored at 2-8°C and protected from light exposure . FITC is susceptible to photobleaching, so limiting light exposure during both storage and experimental procedures is crucial. The antibody is typically formulated in phosphate buffered saline containing <0.1% sodium azide as a preservative . When stored properly, these conjugated antibodies generally maintain activity for at least 12 months, though specific manufacturer recommendations may vary.

What are the validated applications for Goat Anti-Mouse IgG(H+L)-FITC?

Goat Anti-Mouse IgG(H+L)-FITC has been validated for multiple immunological applications. It is quality-tested and proven effective in ELISA/FLISA platforms and flow cytometry . Additional referenced applications include ELISpot, immunohistochemistry (both frozen and paraffin sections), immunocytochemistry, western blot, and multiplex assays . The antibody's versatility makes it valuable for researchers working across different experimental techniques. When using the antibody for a new application, it is advisable to perform appropriate controls to validate its performance in your specific experimental system.

What dilutions are recommended for immunofluorescence and flow cytometry applications?

For immunofluorescence (IF) and immunocytochemistry (ICC) applications, recommended dilutions typically range from 1:100 to 1:500 of the stock antibody solution . For flow cytometry applications, a higher concentration is generally used, with recommended dilutions ranging from 1:50 to 1:200 . These dilution recommendations provide a starting point; optimal dilutions should be determined empirically for each experimental setup. Factors influencing optimal dilution include the abundance of target antigens, the affinity of the primary antibody, and the sensitivity of the detection system.

How can I minimize background fluorescence when using Goat Anti-Mouse IgG(H+L)-FITC in tissue sections?

To minimize background fluorescence when using Goat Anti-Mouse IgG(H+L)-FITC on tissue sections, several strategies are recommended:

• Use an appropriate blocking solution (typically 5-10% normal goat serum) before primary antibody application to reduce non-specific binding.

• Consider using cross-adsorbed secondary antibodies, such as Goat Anti-Mouse IgG, Human ads-FITC, which has been adsorbed against mouse IgM, IgA, and human immunoglobulins to minimize cross-reactivity .

• Implement signal enhancers like Image-iT® FX signal enhancer prior to primary antibody incubation, which has been shown to increase label specificity and resolution .

• Thoroughly wash samples between steps to remove unbound antibodies.

• Use appropriate antifade mounting media such as ProLong® Gold, which has demonstrated enhanced resistance to photobleaching compared to simple buffer solutions .

How can I address photobleaching issues with FITC-conjugated antibodies?

FITC is known to be susceptible to photobleaching, which can significantly impact experimental outcomes, especially in time-lapse imaging or quantitative analysis. To address photobleaching issues:

• Use antifade mounting media such as ProLong® Gold antifade reagent, which has been shown to significantly reduce photobleaching compared to standard phosphate-buffered saline. Evidence shows that samples mounted in ProLong® Gold maintain fluorescence intensity for much longer during continuous illumination with a standard 100-watt Hg-arc lamp .

• Minimize exposure to excitation light during sample preparation and microscopy.

• For critical applications requiring extended imaging periods, consider alternative fluorophores with greater photostability than FITC.

• Store prepared slides at 2-8°C in the dark to slow photobleaching between imaging sessions.

• Consider using newer anti-photobleaching reagents that specifically protect fluorescein-based dyes.

What controls should be included when using Goat Anti-Mouse IgG(H+L)-FITC in multicolor flow cytometry?

When using Goat Anti-Mouse IgG(H+L)-FITC in multicolor flow cytometry, several essential controls should be included:

• Unstained control: Cells without any antibody to establish autofluorescence levels.

• Secondary antibody-only control: Cells stained with only the Goat Anti-Mouse IgG(H+L)-FITC to assess non-specific binding and background.

• Isotype control: Cells stained with an irrelevant mouse antibody of the same isotype as your primary antibody, followed by the FITC-conjugated secondary antibody (using Goat IgG-FITC as an isotype control for the secondary antibody itself can also be valuable) .

• Compensation controls: When using multiple fluorochromes, single-color controls to correct for spectral overlap between fluorophores.

• Fluorescence-minus-one (FMO) controls: Samples stained with all fluorochromes except FITC to establish proper gating strategies.

• Positive and negative biological controls: Samples known to express or not express the target of interest.

How do I determine the optimal concentration of Goat Anti-Mouse IgG(H+L)-FITC for my specific experimental setup?

Determining the optimal concentration of Goat Anti-Mouse IgG(H+L)-FITC for a specific experimental setup requires systematic titration:

• Prepare a dilution series of the secondary antibody (typically ranging from 1:50 to 1:1000) while keeping the primary antibody concentration constant.

• Analyze signal-to-noise ratio at each dilution by comparing signal intensity in positive samples to background in negative controls.

• For quantitative assays like ELISA or FLISA, create standardized curves using purified mouse immunoglobulins as targets. Research has documented this approach where FLISA plates coated with purified mouse IgG, IgM, and IgA were probed with serially diluted Goat Anti-Mouse IgG(H+L)-FITC .

• For cellular applications, assess both signal intensity and specificity at different concentrations through microscopy or flow cytometry.

• The optimal concentration is one that provides maximum specific signal with minimal background staining.

How does cross-adsorption affect the performance of Goat Anti-Mouse IgG(H+L)-FITC in human tissue samples?

Cross-adsorption significantly impacts the performance of Goat Anti-Mouse IgG(H+L)-FITC in human tissue samples by reducing unwanted cross-reactivity:

• Non-adsorbed Goat Anti-Mouse IgG(H+L)-FITC may cross-react with human immunoglobulins and other proteins, resulting in high background staining in human tissues .

• Human-adsorbed versions (e.g., Goat Anti-Mouse IgG, Human ads-FITC) undergo additional purification where the antibody is adsorbed against human immunoglobulins and pooled sera, dramatically reducing cross-reactivity with human proteins .

• The human-adsorbed versions maintain reactivity with mouse IgG heavy chains while minimizing unwanted binding to human components, making them ideal for immunohistochemistry, immunocytochemistry, and flow cytometry applications involving human samples .

• For humanized mouse models or xenograft studies where both mouse and human tissues are present, these cross-adsorbed versions provide superior discrimination between target and non-target immunoglobulins.

• The specificity enhancement comes with a trade-off: human-adsorbed antibodies typically cost more than non-adsorbed versions ($116 vs. $70 for comparable products) .

What considerations are important when using Goat Anti-Mouse IgG(H+L)-FITC in multiplex immunoassays?

When using Goat Anti-Mouse IgG(H+L)-FITC in multiplex immunoassays, several important considerations must be addressed:

• Spectral compatibility: FITC has excitation/emission peaks at approximately 491nm/516nm , which must be compatible with other fluorophores in the panel to minimize spectral overlap.

• Antibody cross-reactivity: When multiple primary antibodies from different species are used, ensure that each secondary antibody is specific to its intended primary target. Cross-adsorbed versions should be considered to minimize unwanted cross-reactions .

• Signal balancing: FITC typically produces moderate signal intensity compared to other fluorophores. When targets of different abundance are being measured, consider the relative brightness of each fluorophore and adjust antibody concentrations accordingly.

• Sequential staining: For complex multiplex panels, sequential staining protocols may be necessary to avoid steric hindrance between antibodies targeting spatially proximal epitopes.

• Referenced applications specifically mention the successful use of Goat Anti-Mouse IgG in multiplex assays, with citations to published studies demonstrating this application .

How do different fixation methods affect the performance of FITC-conjugated antibodies in immunohistochemistry and immunocytochemistry?

Different fixation methods significantly impact the performance of FITC-conjugated antibodies in immunohistochemical applications:

• Paraformaldehyde/formaldehyde fixation: Commonly used and generally compatible with FITC detection, but can create autofluorescence, particularly in tissues rich in elastic fibers or with high aldehyde-reactive amine content. Published research has successfully used FITC-conjugated Goat Anti-Mouse IgG to detect GFAP in paraffin-embedded sections of rat brain tissues .

• Glutaraldehyde fixation: Creates stronger crosslinking but significantly increases autofluorescence in the green spectrum, potentially interfering with FITC signal detection. Additional blocking or quenching steps may be required.

• Methanol/acetone fixation: Provides good preservation of morphology with less autofluorescence than aldehyde-based fixatives, potentially improving signal-to-noise ratio for FITC detection.

• Antigen retrieval methods: Heat-induced or enzymatic antigen retrieval may be necessary for formalin-fixed, paraffin-embedded tissues but can sometimes degrade FITC fluorescence if performed after antibody application.

• Signal enhancement: Techniques like using Image-iT® FX signal enhancer prior to antibody incubation have been demonstrated to improve FITC signal specificity and resolution in fixed cells .