MC4R Antibody, FITC conjugated

What is MC4R and what is its biological significance?

The melanocortin 4 receptor (MC4R) is a member of the melanocortin receptor family, which comprises seven-transmembrane G protein-coupled proteins that activate adenylyl cyclase. MC4R is primarily expressed in the brain and plays crucial roles in energy homeostasis and weight regulation. The melanocortin family includes Alpha-, Beta- and Gamma-melanocyte stimulating hormones (MSH) and adrenocorticotrophin as ligands . MC4R is encoded by the MC4R gene in humans with a predicted protein length of 332 amino acids and a molecular mass of approximately 36.9 kDa . MC4R's subcellular localization is predominantly in the cell membrane, where it functions as a receptor for melanocortins . Understanding MC4R is essential for research in obesity, energy metabolism, and related neurological functions.

What are the benefits of using FITC-conjugated MC4R antibodies in research applications?

FITC (Fluorescein isothiocyanate)-conjugated MC4R antibodies offer several methodological advantages in research contexts:

• Direct visualization without secondary antibodies, reducing background and cross-reactivity issues

• Compatibility with immunofluorescence techniques including IHC-P (paraffin sections), IHC-F (frozen sections), and ICC (immunocytochemistry)

• Enhanced sensitivity for detecting low-abundance MC4R expression

• Streamlined experimental workflows with fewer incubation steps

• Compatibility with multiplex immunofluorescence studies when combined with antibodies conjugated to spectrally distinct fluorophores

The direct conjugation eliminates potential species cross-reactivity problems that can occur with secondary antibodies, making these reagents particularly valuable when working with complex tissue samples or when performing co-localization studies.

What species reactivity can researchers expect from commercially available MC4R antibodies?

Commercial MC4R antibodies demonstrate varied species reactivity profiles. Most commonly available MC4R antibodies, including FITC-conjugated versions, show cross-reactivity with human, mouse, and rat MC4R proteins, making them versatile for comparative studies across these species . Some antibodies have expanded reactivity including primate species such as gibbon, chimpanzee, gorilla, and orangutan . The cross-reactivity stems from the relatively high conservation of MC4R sequence across mammals. When selecting an antibody for a specific research application, it is critical to verify the documented species reactivity and, if possible, validate the antibody in your specific experimental system before proceeding with extensive studies.

What are the validated applications for MC4R antibody, FITC conjugated?

MC4R antibody, FITC conjugated has been validated for several immunological techniques. According to available product information, the primary applications include:

• Immunofluorescence on paraffin-embedded tissue sections (IF/IHC-P)

• Immunofluorescence on frozen tissue sections (IF/IHC-F)

• Immunocytochemistry (ICC)

Some MC4R antibodies are also validated for additional applications, including:

• Enzyme-linked immunosorbent assay (ELISA)

• Immunoprecipitation (IP)

• Western blotting (WB)

The recommended dilution ranges for immunofluorescence applications typically fall within 1:50-1:200 . When adapting a FITC-conjugated MC4R antibody to a novel application, researchers should perform thorough validation studies with appropriate positive and negative controls.

How can researchers optimize immunofluorescence staining protocols using MC4R antibody, FITC conjugated?

Optimizing immunofluorescence protocols with MC4R antibody, FITC conjugated requires systematic approach to several variables:

• Fixation optimization: Test different fixation methods (4% paraformaldehyde, acetone, or methanol) to determine which best preserves MC4R epitopes while maintaining tissue architecture.

• Antigen retrieval methods: For paraffin sections, compare citrate buffer (pH 6.0), EDTA buffer (pH 9.0), and enzymatic retrieval to identify optimal epitope exposure conditions.

• Blocking strategy: Use species-appropriate normal serum (5-10%) with 0.1-0.3% Triton X-100 for permeabilization. For tissues with high background, consider adding 1% BSA and 0.05% Tween-20.

• Antibody titration: Test serial dilutions between 1:50-1:200 to determine optimal signal-to-noise ratio . Starting with manufacturer recommendations, create a dilution series and evaluate staining intensity versus background.

• Incubation conditions: Compare overnight incubation at 4°C versus 1-2 hours at room temperature to optimize signal development.

• Counterstaining considerations: When using FITC-conjugated antibodies (green fluorescence), select counterstains in red or blue spectrum (such as DAPI for nuclei) to avoid spectral overlap.

• Mounting media selection: Use anti-fade mounting media specifically formulated to preserve FITC fluorescence, as FITC is susceptible to photobleaching.

Document all optimization steps methodically to establish a reproducible protocol for future experiments.

What control samples are essential when working with MC4R antibody, FITC conjugated?

Implementing a comprehensive set of controls is critical for interpreting experiments using MC4R antibody, FITC conjugated:

Positive controls:

• Brain tissue sections (particularly hypothalamus) from the same species as your experimental samples, where MC4R is known to be expressed

• Cell lines with validated MC4R expression

• Recombinant cell systems overexpressing MC4R

Negative controls:

• Peptide competition/absorption controls using the immunizing peptide to confirm binding specificity

• MC4R knockout or knockdown tissues/cells where available

• Primary antibody omission controls to assess background autofluorescence

• Isotype controls using non-specific IgG of the same species and concentration as the primary antibody

Procedural controls:

• Tissue sections known to lack MC4R expression

• Sequential dilution series to demonstrate staining concentration-dependence

• Multi-channel fluorescence controls to rule out bleed-through from other fluorophores in multiplex experiments

Systematic use of these controls allows for confident interpretation of MC4R localization and expression patterns in experimental samples.

How do researchers validate the specificity of MC4R antibody, FITC conjugated?

Validating MC4R antibody specificity requires multiple complementary approaches:

• Western blot verification: Though FITC-conjugated antibodies are typically optimized for fluorescence applications, parallel validation with the unconjugated version of the same antibody clone using Western blot can verify that the antibody recognizes a protein of the expected molecular weight (approximately 36.9 kDa for MC4R) .

• Peptide competition assays: Pre-incubating the antibody with the immunizing peptide should abolish or significantly reduce specific staining in both immunoblotting and immunofluorescence applications.

• Correlation with mRNA expression: Use in situ hybridization or RT-PCR to confirm that MC4R protein detection correlates with mRNA expression patterns in tissues.

• Genetic models: Test the antibody in MC4R knockout or knockdown models, which should show reduced or absent staining compared to wild-type samples.

• Cross-validation with multiple antibodies: Compare staining patterns using independent antibodies that recognize different epitopes of MC4R.

• Subcellular localization assessment: Confirm that the staining pattern is consistent with the expected cell membrane localization of MC4R .

• Cross-species reactivity testing: Verify reactivity across specified species (human, mouse, rat) to confirm antibody performance matches manufacturer claims .

Thorough validation enhances confidence in experimental results and helps prevent misinterpretation of staining artifacts.

What sample preparation methods are optimal for MC4R detection using fluorescence microscopy?

Optimal sample preparation for MC4R detection varies by sample type, but these methodological guidelines apply across systems:

For tissue sections (paraffin-embedded):

• Fix tissues in 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin following standard histological procedures

• Section at 4-6 μm thickness

• Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Permeabilize with 0.1-0.3% Triton X-100 to facilitate antibody access to membrane proteins

For frozen tissue sections:

• Flash-freeze tissue in OCT compound using liquid nitrogen-cooled isopentane

• Cryosection at 8-12 μm thickness

• Fix briefly in cold acetone or 4% paraformaldehyde

• Gentle permeabilization may be necessary with 0.1% Triton X-100

For cultured cells:

• Grow cells on glass coverslips or chamber slides

• Fix with 4% paraformaldehyde for 15-20 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for 5-10 minutes

• Block with 5% normal serum from the same species as the secondary antibody

For all sample types, maintain consistent preparation methods across experimental groups to ensure comparable staining intensity. The MC4R antibody, FITC conjugated, can then be applied following manufacturer's recommended dilutions (typically 1:50-1:200) in blocking buffer.

How should researchers determine the appropriate dilution for MC4R antibody, FITC conjugated?

Determining the optimal antibody dilution requires systematic titration to balance specific signal with background:

• Initial range assessment: Start with the manufacturer's recommended dilution range (typically 1:50-1:200 for immunofluorescence applications) .

• Dilution series preparation: Prepare a minimum of 4-5 dilutions spanning and extending beyond the recommended range (e.g., 1:25, 1:50, 1:100, 1:200, 1:400).

• Control inclusion: For each dilution, include both positive control tissue (known to express MC4R) and negative control tissue (lacking MC4R expression).

• Systematic evaluation:

  • Score signal intensity on a scale (0-4+)

  • Assess background fluorescence levels

  • Calculate signal-to-noise ratio for each dilution

  • Document with standardized image acquisition settings

• Verification across sample types: If working with multiple tissue types or preparation methods, verify that the optimal dilution works consistently across all experimental materials.

• Batch testing: For longitudinal studies, test antibody performance across different lots if possible, as conjugation efficiency may vary between manufacturing batches.

The optimal dilution provides robust specific staining with minimal background. For quantitative studies, select a dilution in the linear range of the signal-intensity curve to ensure that signal intensity correlates with actual protein abundance.

Can MC4R antibody, FITC conjugated be used effectively in multiplex immunofluorescence staining?

Yes, MC4R antibody, FITC conjugated can be incorporated into multiplex immunofluorescence protocols with careful experimental design:

Key considerations for successful multiplexing:

• Spectral compatibility: FITC emits green fluorescence (peak ~520 nm), so companion fluorophores should have minimal spectral overlap. Compatible options include:

  • TRITC or Cy3 (red)

  • Cy5 or Alexa Fluor 647 (far red)

  • DAPI or Hoechst (blue)

• Antibody host species combinations: When combining with other primary antibodies, select those raised in different host species to prevent cross-reactivity. Since MC4R antibody, FITC conjugated is typically rabbit-hosted , consider companion antibodies from mouse, goat, or chicken hosts.

• Sequential staining approach: For challenging combinations, apply antibodies sequentially with an intermediate blocking step using excess unconjugated Fab fragments against the first primary antibody species.

• Validation of multiplex combinations: Always validate multiplex staining against single-stain controls to ensure that:

  • Staining patterns remain consistent in multiplex versus single-stain conditions

  • No unexpected cross-reactivity occurs

  • Signal intensity is not diminished by the presence of other antibodies

• Order of application: In some cases, the sequence of antibody application matters. Test different staining sequences if initial results are suboptimal.

Multiplex staining with MC4R antibody, FITC conjugated enables co-localization studies with other proteins of interest, providing valuable insights into functional relationships in complex tissue environments.

How should researchers interpret MC4R staining patterns in different cellular compartments?

Interpretation of MC4R staining patterns requires careful consideration of expected localization and potential artifacts:

Expected MC4R localization patterns:

• Cell membrane predominance: As a G protein-coupled receptor, MC4R primarily localizes to the plasma membrane . Strong membrane staining that outlines cell boundaries represents the canonical pattern.

• Perinuclear/Golgi staining: Some staining in perinuclear regions may represent newly synthesized receptor in the secretory pathway. This pattern may be more prominent in cells with high expression levels.

• Endosomal vesicles: Punctate cytoplasmic staining may indicate receptors undergoing internalization following agonist binding or as part of normal receptor turnover.

Interpretation guidelines:

• Cell-type specific patterns: In brain tissue, MC4R expression is expected primarily in specific neuronal populations. Pattern interpretation should consider the known cell-type distribution of MC4R.

• Correlating with physiological state: MC4R trafficking and surface expression can be regulated by physiological stimuli. Consider how experimental conditions might affect receptor localization.

• Distinguishing artifacts from biology:

  • Uniform cytoplasmic staining often represents non-specific binding

  • Nuclear staining is generally unexpected for MC4R and may indicate antibody cross-reactivity

  • Edge artifacts in tissue sections should not be confused with membrane localization

• Quantitative considerations: For quantitative analysis, distinguish between changes in expression level versus alterations in subcellular distribution.

Compare staining patterns with published literature on MC4R localization and always interpret results in conjunction with appropriate controls to accurately distinguish biological significance from technical artifacts.

What quantitative methods are recommended for analyzing MC4R expression using immunofluorescence data?

Quantitative analysis of MC4R immunofluorescence requires rigorous methodological approaches:

Image acquisition considerations:

• Use consistent microscope settings (exposure time, gain, offset) across all experimental groups

• Capture images below saturation to ensure signal linearity

• Include fluorescence calibration standards if absolute intensity measurements are needed

Quantification methodologies:

• Intensity-based measurements:

  • Mean fluorescence intensity (MFI) within regions of interest (ROIs)

  • Integrated density (area × mean intensity)

  • Background-subtracted intensity using adjacent negative regions

• Distribution analysis:

  • Membrane-to-cytoplasm intensity ratio to assess receptor internalization

  • Line profile analysis across cellular compartments

  • Pearson's or Mander's coefficients for co-localization with other markers

• Population analysis:

  • Percentage of MC4R-positive cells in heterogeneous populations

  • Classification of expression patterns (e.g., membrane-predominant, internalized)

  • Histogram analysis of expression levels across cell populations

Software tools:
Multiple image analysis platforms can be used, including:

• ImageJ/FIJI with appropriate plugins

• CellProfiler for automated high-throughput analysis

• Commercial microscopy software packages

Statistical approaches:

• Compare multiple fields per sample (minimum 5-10)

• Analyze sufficient cell numbers for statistical power (typically >100 cells per condition)

• Apply appropriate statistical tests based on data distribution

• Consider multilevel analysis for nested experimental designs

Rigorous quantification enhances the reproducibility and interpretability of MC4R expression studies using FITC-conjugated antibodies.

How can researchers troubleshoot non-specific binding with MC4R antibody, FITC conjugated?

Non-specific binding is a common challenge with immunofluorescence. Systematic troubleshooting includes:

Common sources of non-specific binding:

• Insufficient blocking: Increase blocking duration (1-2 hours) and consider different blocking agents:

  • 5-10% normal serum from the same species as the secondary antibody

  • 1-3% BSA supplementation

  • Commercial blocking solutions with proprietary formulations

• Over-fixation: Excessive fixation can create artificial epitopes. Optimize fixation duration and consider gentler fixatives for sensitive epitopes.

• Autofluorescence sources:

  • Lipofuscin in aged tissues (can be reduced with Sudan Black B treatment)

  • Formaldehyde-induced autofluorescence (quench with sodium borohydride)

  • Endogenous fluorescent proteins or metabolites

• Antibody concentration: Excessive antibody concentration increases non-specific binding. Repeat titration experiments focusing on more dilute preparations.

Methodological solutions:

• Protocol modifications:

  • Add 0.05-0.1% Tween-20 to washing and antibody dilution buffers

  • Increase washing duration and number of washes

  • Consider low-detergent antibody diluents for reduced background

• Sample-specific treatments:

  • For high-lipid tissues, delipidation steps may reduce background

  • For tissues with high endogenous biotin, use avidin/biotin blocking kits

  • For tissues with high endogenous peroxidase, add quenching steps

• Alternative detection strategies:

  • If persistent issues occur with direct FITC conjugates, consider using unconjugated primary antibody with optimized secondary detection systems

• Validation experiments:

  • Compare staining pattern to alternative MC4R antibodies or detection methods

  • Correlate protein detection with mRNA expression data

  • Implement genetic controls (knockdown/knockout) where available

Systematic troubleshooting with appropriate controls allows researchers to distinguish specific MC4R staining from technical artifacts.

How can MC4R antibody, FITC conjugated be utilized in flow cytometry applications?

While primarily validated for microscopy applications, MC4R antibody, FITC conjugated can be adapted for flow cytometry with these methodological considerations:

Protocol adaptation for flow cytometry:

• Cell preparation:

  • Dissociate tissues to single-cell suspensions using enzymatic methods that preserve surface epitopes

  • For cultured cells, use gentle cell dissociation reagents rather than harsh trypsinization

  • Maintain samples at 4°C during processing to prevent receptor internalization

• Fixation and permeabilization:

  • For total MC4R analysis: Fix with 2-4% paraformaldehyde and permeabilize with 0.1% saponin

  • For surface-only MC4R analysis: Stain live cells or use very gentle fixation without permeabilization

• Antibody labeling:

  • Titrate antibody specifically for flow cytometry applications (optimal concentrations may differ from microscopy)

  • Typical starting dilutions range from 1:50-1:200 based on immunofluorescence recommendations

  • Include FcR blocking reagent to prevent non-specific binding to Fc receptors

• Crucial controls:

  • Fluorescence-minus-one (FMO) controls

  • Isotype controls matched to antibody concentration

  • Positive controls (cells known to express MC4R)

  • Negative controls (cells lacking MC4R expression)

• Data acquisition considerations:

  • Use 488 nm laser excitation for FITC

  • Set compensation if multiplexing with other fluorophores

  • Collect sufficient events (minimum 10,000) for statistical analysis

• Analysis approaches:

  • Quantify percentage of MC4R-positive cells

  • Measure mean/median fluorescence intensity to assess expression levels

  • Consider ratio of surface to total MC4R for trafficking studies

This application extends the utility of MC4R antibody, FITC conjugated beyond traditional microscopy, enabling quantitative analysis of MC4R expression at the single-cell level.

What are the current challenges in MC4R detection and quantification in research contexts?

Researchers face several challenges when studying MC4R using immunological methods:

Technical challenges:

• Antibody specificity issues: G protein-coupled receptors like MC4R share structural similarities, creating potential for cross-reactivity with related receptors like MC3R, MC5R.

• Low expression levels: In many tissues, MC4R expression is relatively low, requiring highly sensitive detection methods and careful signal amplification.

• Conformational epitopes: MC4R has multiple transmembrane domains, and antibodies may recognize conformation-dependent epitopes that are sensitive to sample preparation methods.

• Post-translational modifications: Various states of glycosylation or phosphorylation may affect antibody recognition, complicating interpretation of expression data.

Experimental design challenges:

• Distinguishing receptor variants: MC4R has reported variants and mutations associated with obesity and other conditions, but most antibodies cannot distinguish these variants.

• Receptor trafficking dynamics: MC4R undergoes regulated trafficking between surface and intracellular compartments, complicating interpretation of expression patterns.

• Tissue heterogeneity: In complex tissues like brain, MC4R expression is restricted to specific cell populations, requiring cell-type markers for proper characterization.

Emerging solutions:

• Development of epitope-tagged MC4R constructs for exogenous expression systems

• Implementation of proximity ligation assays to detect MC4R interactions with higher specificity

• Integration of multiple detection methodologies to cross-validate findings

• Application of super-resolution microscopy techniques to better resolve membrane localization

Acknowledging these challenges allows researchers to implement appropriate controls and interpretative caution in MC4R studies.

How does MC4R antibody performance compare across different experimental systems?

MC4R antibody performance varies across experimental systems, necessitating system-specific validation:

Comparative performance table:

Performance variation factors:

• Epitope accessibility: Sample preparation methods significantly impact epitope exposure, with fresh frozen samples typically providing better epitope preservation than fixed samples.

• Expression level differences: Endogenous MC4R expression is often lower than in overexpression systems, requiring more sensitive detection methods for natural tissues.

• Species-specific considerations: Though MC4R antibodies often cross-react with human, mouse, and rat proteins , subtle performance differences may exist across species.

• Background tissue properties: Tissues with high lipid content (like brain) may show different background characteristics than cell lines, necessitating protocol adjustments.

Researchers should validate MC4R antibody, FITC conjugated in their specific experimental system before conducting comprehensive studies, even if the antibody has been validated in other contexts.