Glp1r Antibody, FITC conjugated

What is GLP-1R and why is it an important research target?

GLP-1R (Glucagon-like peptide-1 receptor) is a critical therapeutic target for type 2 diabetes mellitus and metabolic disorders. This receptor actively contributes to maintaining glucose homeostasis while promoting both β cell proliferation and insulin release. The impact of GLP-1R agonists such as semaglutide extends beyond diabetes control, playing multifaceted roles in regulating blood glucose levels, reducing hunger, moderating food intake, and managing body weight. Recent research has also indicated that GLP-1R agonists may inhibit cancer progression in some malignant tumors .

At the molecular level, GLP-1R's activity is mediated by G proteins which activate adenylyl cyclase, leading to downstream signaling cascades that regulate insulin secretion and other metabolic processes. Understanding GLP-1R localization and function is therefore essential for developing targeted therapies for metabolic disorders .

What are the key specifications of FITC-conjugated GLP-1R antibodies available for research?

FITC-conjugated GLP-1R antibodies are available with various specifications optimized for different experimental applications. Common specifications include:

When selecting an antibody for your research, consider these specifications in relation to your specific experimental requirements and the biological system you're investigating.

What are the optimal dilution ranges for different applications of FITC-conjugated GLP-1R antibody?

Optimal dilution ranges vary significantly depending on the application. Based on available technical data, the following dilution ranges are recommended:

It's strongly recommended to perform a dilution series experiment to determine the optimal concentration for your specific experimental conditions, tissue type, and expression level of GLP-1R in your samples.

How can I validate the specificity of a GLP-1R antibody in my experimental system?

Validating antibody specificity is crucial for generating reliable results. Current literature suggests several approaches:

• Genetic validation: Use GLP-1R knockout tissues/cells as negative controls. For human tissues where knockouts are not readily available, CRISPR deletion or stable lentiviral shRNA knockdown in cell lines like EndoC-βH should be considered .

• Comparison with alternative detection methods: Compare antibody labeling with fluorescently labeled GLP-1R agonists such as exendin-4 conjugates (Ex4-Cy3, Ex4-Cy5) or liraglutide/semaglutide conjugates .

• Peptide competition assays: Pre-incubate the antibody with excess immunizing peptide to confirm signal reduction.

• Multiple antibody validation: Use multiple antibodies targeting different epitopes of GLP-1R and confirm consistent staining patterns.

• Cell line controls: Use human-derived cell lines transfected with and without human GLP-1R, or with graded expression levels (low and high) .

Remember that only a few GLP-1R antibodies have been demonstrated to be truly specific. According to current literature, validated monoclonal antibodies include Glp1R0017, Mab 7F38, and GLP1R-APC, which have been rigorously tested against knockout tissues .

What are the best tissue preparation methods for GLP-1R immunofluorescence staining?

Proper tissue preparation is critical for successful GLP-1R immunofluorescence staining:

• Fixation:

  • For cultured cells: 4% paraformaldehyde for 10-15 minutes at room temperature

  • For tissue sections: 4% paraformaldehyde, either through perfusion or immersion fixation for 24-48 hours

  • Note: Overfixation can mask epitopes; optimize fixation time for your specific tissue

• Antigen retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • For many GLP-1R epitopes, citrate buffer works well, but optimization may be necessary

• Permeabilization:

  • For membrane receptors like GLP-1R, gentle permeabilization with 0.1-0.3% Triton X-100 is recommended

  • Over-permeabilization can disrupt membrane structure and epitope accessibility

• Blocking:

  • Use 5-10% normal serum (from the same species as the secondary antibody) with 1% BSA

  • Include 0.1% Tween-20 to reduce background

• Tissue thickness:

  • For confocal microscopy: 5-20 μm sections are optimal

  • For widefield fluorescence: thinner sections (5-10 μm) often yield better results

When working with pancreatic tissue, which is rich in proteases, add protease inhibitors to all solutions and process tissues quickly to preserve antigenicity.

How can I effectively investigate GLP-1R internalization dynamics using FITC-conjugated antibodies?

Investigating GLP-1R internalization dynamics requires careful experimental design:

• Live cell imaging approach:

  • Use antagonistic FITC-conjugated GLP-1R antibodies that don't trigger internalization themselves

  • Pulse-label surface receptors at 4°C (to prevent internalization)

  • Wash extensively to remove unbound antibody

  • Shift to 37°C and add GLP-1R agonist (e.g., GLP-1, exendin-4)

  • Image at selected time points using confocal microscopy

• Fixed cell approach for quantitative assessment:

  • Treat cells with agonist for various time points

  • Fix and label with FITC-conjugated GLP-1R antibody

  • Counterstain with markers for early endosomes (EEA1) and late endosomes/lysosomes (LAMP1)

  • Quantify colocalization at different time points

Recent research has shown that bispecific molecules like GIPR-Ab/GLP-1 induce internalization of both GLP-1R and GIPR, accompanied by amplified cAMP production . This suggests that receptor internalization dynamics may be particularly important for understanding the efficacy of multi-target therapeutic approaches.

When analyzing internalization, consider:

• Rate of internalization (% internalized over time)

• Route of internalization (clathrin-dependent vs. independent)

• Receptor recycling vs. degradation rates

• Endosomal signaling (internalized receptors may continue signaling from endosomes)

What are the considerations for dual labeling experiments using FITC-conjugated GLP-1R antibody and other markers?

Dual labeling experiments require careful consideration of several factors:

• Spectral compatibility:

  • FITC excitation/emission (495nm/519nm) must be compatible with other fluorophores

  • Recommended compatible pairs: FITC with Cy3, Texas Red, or Alexa 594/647

  • Avoid spectrally overlapping fluorophores like FITC and GFP

• Antibody cross-reactivity:

  • When using multiple primary antibodies, ensure they're from different host species

  • If using same-species antibodies, sequential immunostaining with complete blocking between steps is necessary

• Control experiments:

  • Single-label controls to assess bleed-through

  • Secondary-only controls to assess non-specific binding

  • Absorption controls with immunizing peptides

• Sample preparation optimization:

  • Some fixation methods may preserve one epitope but damage another

  • Test multiple fixation protocols if dual labeling is unsuccessful

• Analysis considerations:

  • Use proper colocalization analysis (Pearson's or Manders' coefficients)

  • Account for differences in signal intensity between channels

  • Consider 3D analysis for volumetric colocalization in tissue sections

For studying GLP-1R interaction with GIPR, recent research has used sophisticated approaches to detect both receptors simultaneously, revealing that bispecific molecules can bind to GIPR and GLP-1R simultaneously and trigger receptor internalization, amplifying endosomal cAMP signaling in cells expressing both receptors .

How do FITC-conjugated antibodies compare with other methods for detecting GLP-1R in live tissues?

Multiple approaches exist for detecting GLP-1R in tissues, each with distinct advantages and limitations:

Research indicates that each method has unique capabilities. For example, fluorescent agonists like Ex4-Cy3/Cy5 and fluorescently labeled liraglutide/semaglutide have been successfully used to study GLP-1R in pancreatic and brain tissues . Meanwhile, antagonistic antibodies like Glp1R0017 and GLP1R-APC have been shown to effectively enrich islet cells according to their GLP-1R expression level .

When designing your experiment, consider combining multiple detection methods for cross-validation and more comprehensive understanding of receptor biology.

How should quantitative analysis of GLP-1R expression be performed using FITC-labeled antibodies?

Quantitative analysis of GLP-1R expression requires standardized approaches:

• Flow cytometry quantification:

  • Use calibration beads with known fluorophore quantities to generate standard curves

  • Calculate molecules of equivalent soluble fluorochrome (MESF) to standardize between experiments

  • Include isotype controls and unstained samples

  • Consider using median fluorescence intensity (MFI) rather than mean values

  • Apply compensation for spectral overlap when using multiple fluorophores

• Microscopy-based quantification:

  • Use consistent exposure settings between samples

  • Apply background subtraction based on no-primary-antibody controls

  • Consider using internal controls (cells/tissues with known expression) in each experiment

  • For tissue sections, normalize to number of cells or area

  • Use automated analysis software to reduce bias

• Western blot quantification:

  • Include loading controls appropriate for your sample type

  • Generate standard curves using recombinant proteins if absolute quantification is needed

  • Use appropriate normalization (total protein via stain-free gels or housekeeping proteins)

• Addressing variability:

  • Run technical replicates (minimum of 3)

  • Include biological replicates (minimum of 3 independent samples)

  • Report both statistical significance and effect sizes

When comparing between studies or experimental conditions, it's essential to maintain consistent antibody concentrations, exposure times, and image acquisition settings to ensure valid comparisons.

How can I troubleshoot weak or absent GLP-1R staining despite confirmed expression?

Weak or absent GLP-1R staining despite confirmed expression is a common challenge. Consider these troubleshooting approaches:

• Epitope masking issues:

  • Try multiple antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0, enzymatic retrieval)

  • Test different fixation protocols (reduced fixation time, alternative fixatives)

  • For frozen sections, try brief post-fixation in acetone or methanol

• Antibody penetration problems:

  • Increase permeabilization (0.1-0.5% Triton X-100)

  • Extend antibody incubation times (overnight at 4°C)

  • For thick sections, consider clearing techniques (CLARITY, iDISCO)

• Low expression levels:

  • Use signal amplification systems (tyramide signal amplification, polymer detection systems)

  • Increase antibody concentration (though carefully monitor background)

  • Try different GLP-1R antibodies targeting distinct epitopes

• Technical considerations:

  • Check microscope settings (appropriate filter sets, laser power/gain settings)

  • Ensure FITC hasn't photobleached (minimize light exposure during processing)

  • Consider autofluorescence quenching (Sudan Black B, TrueBlack)

• Biological considerations:

  • Confirm GLP-1R expression in your specific tissue/cell type and condition

  • Consider receptor internalization status (surface vs. internalized pools)

  • Check expression timing (GLP-1R levels may fluctuate with metabolic state)

It's worth noting that even EndoC-βH1 human beta cell lines, which mount insulin secretory responses to GLP-1R agonists and express GLP-1R mRNA, sometimes show undetectable GLP-1R protein levels using antibodies or probes .

What are the potential sources of false positives in GLP-1R detection, and how can they be addressed?

Several factors can lead to false positive results when detecting GLP-1R:

• Antibody cross-reactivity:

  • Validate antibody specificity using knockout tissues/cells where available

  • Perform peptide competition assays to confirm binding specificity

  • Compare staining patterns with multiple independent antibodies

  • Current research indicates that many commercial GLP-1R antibodies have variable specificity, and only a few (like Glp1R0017, Mab 7F38) have been extensively validated

• Autofluorescence:

  • Include unstained controls to assess natural tissue autofluorescence

  • Use autofluorescence quenching reagents (Sudan Black B, TrueBlack)

  • Consider spectral unmixing during image acquisition

  • For tissues with high autofluorescence (brain, liver), consider using far-red fluorophores instead of FITC

• Non-specific binding:

  • Optimize blocking conditions (try different blockers: BSA, normal serum, casein)

  • Increase washing duration and frequency

  • Include detergents in washing buffers (0.05-0.1% Tween-20)

  • Test different antibody dilutions to find optimal signal-to-noise ratio

• Fixation artifacts:

  • Compare results across multiple fixation methods

  • Include appropriate controls for each fixation protocol

  • Consider live-cell labeling approaches where feasible

• Image processing artifacts:

  • Be cautious with contrast enhancement during image processing

  • Establish consistent thresholds for quantification

  • Process all experimental and control images identically

Recent literature emphasizes the importance of proper reagent validation. As noted in search result , "Current GLP1R antibodies have variable specificity and, except for the monoclonal antagonistic antibody, can only be used in fixed tissue."

How can FITC-conjugated GLP-1R antibodies be utilized in receptor trafficking studies?

FITC-conjugated GLP-1R antibodies can be valuable tools for studying receptor trafficking when used in sophisticated experimental designs:

• Pulse-chase approaches:

  • Label surface receptors with non-activating FITC-conjugated antibodies

  • Allow internalization to occur naturally or induce with agonists

  • Track receptor movement through endocytic compartments over time

  • Co-label with markers for different cellular compartments (EEA1 for early endosomes, Rab11 for recycling endosomes, LAMP1 for lysosomes)

• Receptor recycling quantification:

  • Label surface receptors with FITC-conjugated antibodies

  • Allow internalization with agonist treatment

  • Strip remaining surface antibodies with mild acid wash

  • Measure reappearance of FITC signal at the membrane over time to quantify recycling

• Compartment-specific co-localization:

  • Use FITC-GLP-1R antibodies with spectrally distinct markers for cellular compartments

  • Perform quantitative co-localization analysis

  • Track receptor progression through the endocytic pathway

• Live-cell super-resolution microscopy:

  • Use FITC-conjugated GLP-1R antibody fragments (Fab) to minimize receptor crosslinking

  • Apply super-resolution techniques (STORM, PALM) for nanoscale visualization

  • Track receptor clustering and organization during activation and internalization

Research has shown that receptor internalization and trafficking are critical aspects of GLP-1R signaling. For instance, bispecific molecules like GIPR-Ab/GLP-1 induce internalization of both GLP-1R and GIPR, which is associated with amplified cAMP production in endosomal compartments . This suggests that understanding receptor trafficking may provide insights into the therapeutic efficacy of GLP-1R-targeting drugs.

What are the considerations for studying GLP-1R in specialized cell types or tissues?

Studying GLP-1R in specialized tissues requires tailored approaches for different biological contexts:

• Pancreatic β-cells:

  • Consider the high proteolytic activity in pancreatic tissue (use protease inhibitors)

  • Account for autofluorescence from insulin granules

  • Co-stain with insulin to specifically identify β-cells

  • GLP-1R expression may vary with metabolic state and diabetes progression

• Brain tissue:

  • Address high lipid content and strong autofluorescence

  • Use thinner sections (10-20 μm) to improve antibody penetration

  • Extended antibody incubation times (24-48 hours) may be necessary

  • Consider antigen retrieval optimization for fixed brain tissue

• Adipose tissue:

  • Address challenges with lipid droplets and autofluorescence

  • Consider delipidation steps during sample preparation

  • Optimize fixation protocols to preserve membrane structures

  • GLP-1R expression in adipose tissue may be lower than in pancreas or brain

• Intestinal L-cells:

  • Co-stain with L-cell markers like GLP-1 or PYY

  • Account for mucus and glycocalyx interference with antibody binding

  • Consider harsh antigen retrieval methods to overcome cross-linking

• Cardiovascular tissues:

  • Address elastic fiber autofluorescence

  • Consider perfusion fixation for better tissue preservation

  • Examine expression in different vascular beds, which may vary considerably

Tissue-specific detection patterns have been reported in the literature. For example, in studies tracking tissue distribution, the rank ordering of GLP-1R detection in tissues for mGIPR-Ab was BAT>pancreas>WAT>brain, while for a bispecific molecule (mGIPR-Ab/P1), it was pancreas>BAT>WAT>brain , highlighting the importance of understanding tissue-specific detection patterns.

How can FITC-conjugated GLP-1R antibodies be combined with other advanced imaging technologies?

Integrating FITC-conjugated GLP-1R antibodies with advanced imaging technologies can provide unprecedented insights:

• Multi-photon microscopy:

  • Enables deeper tissue penetration (up to 1 mm)

  • Reduces photobleaching of FITC

  • Allows for intravital imaging of GLP-1R in live animal models

  • Requires specialized equipment but provides superior resolution in thick tissues

• Super-resolution microscopy:

  • STED (Stimulated Emission Depletion) microscopy can resolve FITC-labeled structures below the diffraction limit (~50 nm)

  • STORM/PALM approaches provide nanoscale resolution of receptor clustering

  • Structured Illumination Microscopy (SIM) offers 2x improvement in resolution with standard fluorophores

• Correlative Light and Electron Microscopy (CLEM):

  • Combine FITC-based fluorescence imaging with electron microscopy

  • Requires specialized sample preparation with EM-compatible fluorescent preservation

  • Provides ultrastructural context for GLP-1R localization

• Light-sheet microscopy:

  • Enables rapid 3D imaging of large tissue volumes

  • Reduces photobleaching compared to confocal microscopy

  • Ideal for developmental studies or whole-organ GLP-1R mapping

• Expansion microscopy:

  • Physically expands specimens using hydrogels

  • Improves effective resolution of conventional microscopes

  • Compatible with standard FITC-conjugated antibodies

  • Particularly useful for crowded cellular compartments

• Intravital imaging:

  • Requires minimally invasive surgical approaches

  • Can track GLP-1R dynamics in living organisms

  • Often combined with genetic reporters for contextual information

When implementing these advanced technologies, it's essential to validate that the FITC signal remains specific to GLP-1R through appropriate controls. Additionally, specialized sample preparation may be required for each technology, and optimization of antibody concentration and incubation conditions may differ from standard protocols.