Goat Anti-Mouse IgG (H&L) - AF555

What is Goat Anti-Mouse IgG (H+L) - AF555 and what are its key specifications?

Goat Anti-Mouse IgG (H+L) - AF555 is a secondary antibody produced in goats that recognizes mouse IgG heavy and light chains and is conjugated to the fluorescent dye Alexa Fluor 555. This reagent is derived from pooled antisera from goats hyperimmunized with mouse IgG and purified through affinity chromatography on mouse IgG covalently linked to agarose . The antibody specifically reacts with the heavy and light chains of mouse IgG1, IgG2a, IgG2b, IgG2c, and IgG3, as well as with the light chains of mouse IgM and IgA . The fluorophore has an excitation maximum of approximately 555 nm and an emission maximum around 565 nm, making it compatible with green (532 nm) or yellow-green (561 nm) lasers .

The standard formulation includes:

• Concentration: 1.0 mg/mL

• Buffer: Phosphate buffered saline containing <0.1% sodium azide

• Clonality: Polyclonal

• Recommended storage: 2-8°C with protection from light exposure

What does the "(H+L)" designation indicate and how does it affect experimental applications?

The "(H+L)" designation indicates that the antibody recognizes both heavy (H) and light (L) chains of mouse IgG molecules. This has several important implications for research applications:

• The antibody will bind to the species-specific heavy chains of mouse IgG as well as the kappa and lambda light chains that are common across different immunoglobulin classes .

• Because light chains are shared between different immunoglobulin isotypes, this antibody may also recognize mouse IgM and IgA through their light chains , which could be important when designing experiments requiring absolute isotype specificity.

• The ability to bind both heavy and light chains potentially increases the signal amplification compared to antibodies that only recognize specific regions, providing enhanced detection sensitivity in most applications.

• When working with samples containing multiple immunoglobulin classes, researchers should consider potential cross-reactivity through the light chain recognition .

What research applications is Goat Anti-Mouse IgG (H+L) - AF555 suitable for?

Based on manufacturer validation data, Goat Anti-Mouse IgG (H+L) - AF555 is suitable for multiple research applications:

For immunocytochemistry applications, manufacturers typically recommend a concentration range of 2.5-5.0 μg/mL . The recommended dilution for immunofluorescence applications generally falls between 1:500-1:2000 . When using this antibody for the first time in a new experimental system, titration experiments should be performed to determine the optimal concentration for your specific application.

How do different cross-adsorption versions of Goat Anti-Mouse IgG (H+L) - AF555 compare?

Multiple versions of Goat Anti-Mouse IgG (H+L) - AF555 with different cross-adsorption profiles are available to meet various experimental needs:

• Standard (non-adsorbed) version:

  • No specific cross-adsorption

  • May react with immunoglobulins from other species

  • Best for simple systems with mouse antibodies on non-immunological samples

• Cross-adsorbed version:

  • Adsorbed against human IgG and human serum

  • Minimizes reactivity with human samples

  • Suitable for mouse antibodies on human tissues

• Multi-species cross-adsorbed version:

  • Adsorbed against human, rat, hamster, goat, sheep, rabbit, chicken, guinea pig, horse, and bovine serum proteins

  • Minimal reactivity with a broad range of species

  • Ideal for complex multi-species systems and multiplexing applications

• Highly cross-adsorbed version:

  • Extensive adsorption against bovine IgG, goat IgG, rabbit IgG, rat IgG, human IgG, and human serum

  • Maximum specificity for mouse IgG

  • Essential for multi-color immunofluorescence with antibodies from multiple species

The choice between these versions depends on your experimental design. For standard single-color immunofluorescence of mouse tissues, the non-adsorbed version may be sufficient. For experiments involving human samples or multiple primary antibodies from different species, select the appropriate cross-adsorbed version to minimize background and cross-reactivity issues.

How should Goat Anti-Mouse IgG (H+L) - AF555 be stored and handled to maintain optimal activity?

Proper storage and handling are critical for maintaining the functionality of Goat Anti-Mouse IgG (H+L) - AF555:

Storage recommendations:

• Temperature: Store between 2-8°C (refrigerated)

• Light protection: Critical to protect from prolonged light exposure to prevent photobleaching

• Avoid freezing: Do not freeze the antibody as this can damage both the protein structure and fluorophore conjugation

• Avoid repeated freeze-thaw cycles: These can lead to protein denaturation and fluorophore degradation

Handling guidelines:

• Centrifuge briefly before use: This helps eliminate any protein aggregates that may have formed during storage and reduces non-specific background staining

• Use only the supernatant after centrifugation to minimize aggregates

• For the fluorophore-labeled antibodies, a final working concentration of 1-10 μg/mL is typically recommended for most immunofluorescence applications

• Prepare fresh working dilutions immediately before use whenever possible

Following these storage and handling guidelines will help maintain the quality and performance of the antibody throughout its expected shelf life, ensuring consistent experimental results.

What is the optimal dilution range for Goat Anti-Mouse IgG (H+L) - AF555 in different applications?

The optimal dilution varies by application type, but manufacturers provide the following general guidelines:

Important methodological considerations:

• The optimal dilution should be determined empirically for each experimental system through titration experiments.

• For immunocytochemistry applications, BioLegend recommends a concentration range of 2.5-5.0 μg/mL .

• Cell Signal recommends a final dilution of 1:500-1:2000 for immunofluorescence applications .

• Factors affecting optimal concentration include target abundance, primary antibody affinity, sample type, and detection system sensitivity.

When titrating, prepare a series of dilutions and determine which provides the optimal signal-to-background ratio for your specific application. Each new experimental system (new tissue type, fixation method, or detection system) may require re-optimization.

How can I minimize background fluorescence when using Goat Anti-Mouse IgG (H+L) - AF555?

Minimizing background fluorescence is crucial for obtaining clean, interpretable results with Goat Anti-Mouse IgG (H+L) - AF555:

• Antibody selection strategies:

  • Use cross-adsorbed or highly cross-adsorbed versions when working with tissues containing endogenous immunoglobulins

  • Consider F(ab')2 fragments to eliminate Fc-mediated background when working with Fc receptor-expressing cells

  • Select multi-species adsorbed antibodies for complex samples containing proteins from multiple species

• Protocol optimization:

  • Centrifuge the antibody solution briefly before use to remove aggregates that can cause non-specific binding

  • Use only the supernatant after centrifugation in your experiments

  • Include appropriate blocking steps with 5-10% serum from the same species as the secondary antibody

  • Optimize washing steps (increase number and duration of washes)

  • Titrate the secondary antibody to find the minimum concentration that provides adequate signal

• Sample-specific considerations:

  • For mouse tissues: Use M.O.M. (Mouse-on-Mouse) blocking kits to reduce endogenous mouse IgG detection

  • For highly autofluorescent tissues: Consider spectral unmixing or longer wavelength fluorophores

  • For samples with endogenous biotin: Include avidin/biotin blocking steps if using biotin-based detection systems

• Controls to include:

  • Secondary-only control (omit primary antibody) to assess non-specific binding

  • Isotype control to evaluate primary antibody specificity

  • Unstained sample to assess autofluorescence levels

These strategies should be applied systematically to identify and address the specific sources of background in your experimental system.

What are the critical factors to consider when designing multiplexed immunofluorescence experiments using Goat Anti-Mouse IgG (H+L) - AF555?

Designing successful multiplexed immunofluorescence experiments with Goat Anti-Mouse IgG (H+L) - AF555 requires careful consideration of several factors:

• Fluorophore compatibility:

  • Alexa Fluor 555 has an excitation maximum of approximately 555 nm and emission maximum around 565 nm

  • Pair with fluorophores that have minimal spectral overlap (e.g., Alexa Fluor 488 and Alexa Fluor 647)

  • Consider the excitation lasers and emission filters available on your imaging system

  • Alexa Fluor 555 is optimally excited by green (532 nm) or yellow-green (561 nm) lasers

• Cross-reactivity prevention:

  • Use highly cross-adsorbed secondary antibodies to prevent cross-detection between species

  • The multi-species adsorbed variant (specifically adsorbed against human, rat, hamster, goat, sheep, rabbit, chicken, guinea pig, horse, and bovine serum proteins) is ideal for complex multi-labeling experiments

  • Consider the host species of all primary antibodies in your multiplexing panel

• Sequential staining strategies:

  • For complex multiplexing, consider sequential staining with intermediate fixation steps

  • Blocking between sequential staining rounds can minimize cross-reactivity

  • Use directly conjugated primary antibodies when possible to reduce species constraints

• Controls for multiplexing:

  • Single-color controls for spectral compensation/unmixing

  • FMO (Fluorescence Minus One) controls to establish gating boundaries

  • Absorption controls to confirm specificity of each antibody in the presence of others

By carefully addressing these considerations, researchers can develop robust multiplexed immunofluorescence protocols that provide reliable and reproducible results.

How do F(ab')2 fragments of Goat Anti-Mouse IgG-AF555 compare to whole IgG conjugates for specialized applications?

F(ab')2 fragments offer distinct advantages in certain research contexts compared to whole IgG conjugates:

The Cell Signaling Technology F(ab')2 fragment product specifically notes that it "results in less non-specific binding, as it lacks the Fc domain that can bind to the cells with Fc receptors" . This makes F(ab')2 fragments particularly valuable for:

• Samples containing Fc receptor-expressing cells:

  • Immune tissues rich in macrophages, dendritic cells, B cells

  • Flow cytometry of leukocytes

  • Immunofluorescence of lymphoid tissues

• Application-specific advantages:

  • Reduced background in flow cytometry of immune cells

  • Improved penetration in thick tissue sections

  • Minimized non-specific binding in frozen sections

  • Reduced antibody-induced clustering of target proteins in live cell imaging

When deciding between whole IgG and F(ab')2 fragments, researchers should consider the specific requirements of their experimental system, particularly the presence of Fc receptors and the need for minimal non-specific binding.

What are the potential cross-reactivity issues when using Goat Anti-Mouse IgG (H+L) - AF555 in complex experimental systems?

Understanding potential cross-reactivity is essential for accurate interpretation of results in complex systems:

• Inherent cross-reactivity of non-adsorbed versions:

  • May react with immunoglobulins from other species due to conserved epitopes

  • Cross-reactivity with rat primary antibodies has been observed even in some cross-adsorbed products

  • Light chain recognition may lead to detection of other immunoglobulin classes (IgM, IgA)

• Species-specific considerations:

  • Standard versions may react with endogenous immunoglobulins in tissues

  • Cross-reactivity with rat has been specifically noted in some product descriptions

  • Even highly cross-adsorbed versions may retain some reactivity to closely related species

• Strategies for minimizing cross-reactivity issues:

  • Select the appropriate cross-adsorption level for your experimental system

  • The multi-species specific pre-adsorbed version (adsorbed against human, rat, hamster, goat, sheep, rabbit, chicken, guinea pig, horse, and bovine serum proteins) offers the broadest protection against cross-reactivity

  • For human samples, use antibodies cross-adsorbed against human IgG and human serum

  • For multicolor immunofluorescence involving multiple species, use highly cross-adsorbed secondary antibodies

• Critical controls to assess cross-reactivity:

  • Secondary-only controls on each tissue type to evaluate direct binding

  • Isotype controls to assess non-specific primary antibody binding

  • Absorption controls (pre-incubating secondary with irrelevant IgG)

By carefully selecting the appropriate secondary antibody formulation and including the necessary controls, researchers can minimize cross-reactivity issues and ensure specific detection of their target antigens.

How does the degree of labeling (DOL) affect the performance of Goat Anti-Mouse IgG (H+L) - AF555 in quantitative applications?

The degree of labeling (DOL) refers to the number of fluorophore molecules conjugated to each antibody molecule, which significantly impacts performance in quantitative applications:

Typical DOL specifications:

• Alexa Fluor 555 conjugates typically have 2-8 fluorophore molecules per IgG molecule

• The exact DOL is often indicated on the certificate of analysis for each product lot

Impact on experimental performance:

For quantitative applications:

• Consistency is critical - use the same lot when possible or normalize based on DOL

• Document the DOL information for each experiment to allow for comparison

• For absolute quantification, consider calibration with standards of known fluorophore concentration

• When comparing expression levels between samples, maintain identical staining conditions and acquisition parameters

The high photostability of Alexa Fluor 555 makes it suitable for quantitative applications requiring extended imaging sessions or high laser power, such as confocal microscopy and quantitative image analysis .

What spectral considerations are important when using Goat Anti-Mouse IgG (H+L) - AF555 in combination with other fluorophores?

Spectral properties and compatibility are critical when designing multi-color immunofluorescence experiments:

Alexa Fluor 555 spectral characteristics:

• Excitation maximum: approximately 555 nm

• Emission maximum: approximately 565 nm

• Best excited by: Green laser (532 nm) or Yellow-Green laser (561 nm)

• Can also be excited by: Blue laser (488 nm), though less efficiently

Optimal fluorophore combinations:

Imaging system considerations:

• Filter sets: Use bandpass filters that capture emission around 565-580 nm

• Laser selection: Green (532 nm) or Yellow-Green (561 nm) lasers provide optimal excitation

• Detector sensitivity: PMT or camera sensitivity in the yellow-orange range

• Spectral unmixing: May be necessary for complex multi-fluorophore experiments

Minimizing bleed-through:

• Acquire single-color controls for compensation/unmixing

• Consider sequential acquisition for closely overlapping fluorophores

• Balance signal intensities between different channels

• Use appropriate dichroic mirrors and emission filters

By carefully considering these spectral properties, researchers can design multi-color panels that provide clear separation between fluorophores and minimize bleed-through between channels.

What are the most common causes of high background when using Goat Anti-Mouse IgG (H+L) - AF555 and how can they be addressed?

High background is a common challenge when using fluorescent secondary antibodies. Here are the main causes and solutions:

• Antibody-related factors:

• Sample-specific issues:

• Protocol optimization approaches:

Essential controls to include:

• Secondary antibody-only control (omit primary antibody)

• Isotype control (irrelevant primary antibody of the same isotype)

• Absorption control (pre-incubate primary with excess antigen)

• Unstained control (to assess autofluorescence)

By systematically addressing these factors and including appropriate controls, researchers can significantly reduce background and improve the signal-to-noise ratio in their experiments.

How can I troubleshoot weak or absent signal when using Goat Anti-Mouse IgG (H+L) - AF555?

Weak or absent signal can result from multiple factors in the experimental workflow:

• Antibody-related issues:

• Sample preparation factors:

• Detection and imaging issues:

Methodical troubleshooting approach:

• Test antibody functionality with a simple positive control sample

• Systematically modify one parameter at a time

• Include appropriate controls with each experiment

• Document all experimental conditions and imaging parameters

By following this systematic approach, researchers can identify and address the specific factors causing weak or absent signal in their experiments.

What controls are essential when using Goat Anti-Mouse IgG (H+L) - AF555 in immunofluorescence experiments?

A comprehensive set of controls ensures valid and interpretable results:

• Specificity controls:

• Technical controls:

• Quantification controls:

All controls should be processed alongside experimental samples using identical protocols, reagents, and incubation times. They should be imaged using the same acquisition parameters and analyzed using consistent methods to ensure valid interpretation of results.

How should Goat Anti-Mouse IgG (H+L) - AF555 be optimized for different imaging platforms and techniques?

Optimizing Goat Anti-Mouse IgG (H+L) - AF555 for different imaging platforms requires consideration of the fluorophore's properties and the specific requirements of each technique:

• Widefield fluorescence microscopy:

• Confocal microscopy:

• Flow cytometry:

• Super-resolution techniques:

General optimization principles:

• Always titrate the antibody for each specific application and sample type

• Balance signal intensity with background and photobleaching concerns

• Include appropriate controls for each imaging modality

• Document all parameters for reproducibility

By systematically optimizing these parameters for each imaging platform, researchers can maximize the performance of Goat Anti-Mouse IgG (H+L) - AF555 across a wide range of experimental applications.