GRIN2C Antibody, FITC conjugated

What is the GRIN2C protein and why is it significant in neuroscience research?

GRIN2C (also known as GluN2C or NMDAR2C) is a subunit of the N-methyl-D-aspartate (NMDA) receptor, which belongs to the ionotropic glutamate receptor superfamily. NMDA receptors are critical components of the central nervous system, forming heteromeric assemblies typically composed of GluN1 subunits combined with one or more GluN2 subunits (A-D). These receptors form channels permeable to calcium, potassium, and sodium ions .

GRIN2C has significant research value because:

• It exhibits distinct biophysical properties compared to other NMDA receptor subunits

• It has a specialized expression pattern, with high expression in cerebellar tissues

• Recent studies have identified GRIN2C as abundantly expressed in specific brain regions such as the ventral tegmental area (VTA)

• Alterations in GRIN2C are associated with several neurological and psychiatric conditions including Alzheimer's disease, Parkinson's disease, and schizophrenia

GRIN2C-containing NMDA receptors play crucial roles in regulating excitatory-inhibitory balance in neural circuits, particularly in the medial prefrontal cortex, where they influence excitatory postsynaptic currents, inhibitory postsynaptic currents, and spine density .

How does FITC conjugation enhance the utility of GRIN2C antibodies in experimental research?

FITC (Fluorescein Isothiocyanate) conjugation provides several advantages for GRIN2C antibody applications:

• Direct visualization: FITC conjugation eliminates the need for secondary antibodies, reducing protocol complexity and potential cross-reactivity issues .

• Spectral properties: FITC has an excitation maximum at 499 nm and emission maximum at 515 nm, making it compatible with standard fluorescence microscopy filter sets and the 488 nm laser line commonly found in confocal microscopes and flow cytometers .

• Multiplexing capability: FITC's spectral profile allows it to be used alongside other fluorophores in multi-color immunofluorescence studies, facilitating co-localization experiments with other proteins of interest .

• Quantitative applications: FITC-conjugated antibodies enable quantitative assessment of protein expression through flow cytometry or fluorescence intensity measurements in microscopy applications.

• Stability: When properly stored (protected from light, at -20°C), FITC-conjugated antibodies maintain their fluorescence properties, allowing for consistent experimental results .

The direct conjugation eliminates potential variations in secondary antibody binding, providing more consistent staining results for critical applications such as examining GRIN2C distribution in neural tissues or co-localization with synaptic markers.

What are the main experimental applications for GRIN2C antibody, FITC conjugated?

GRIN2C antibody with FITC conjugation can be employed in multiple experimental approaches:

Immunofluorescence (IF) microscopy:

• Visualization of GRIN2C expression patterns in fixed tissues or cultured cells

• Recommended dilutions typically range from 1:50-1:200, though optimal concentration should be determined empirically for each application

• Particularly useful for examining subcellular localization within neurons, including synaptic localization

Immunohistochemistry (IHC):

• Detection of GRIN2C in tissue sections, allowing assessment of expression patterns across brain regions

• Typically used at dilutions of 1:20-1:200

• Useful for comparative studies examining changes in GRIN2C distribution in disease models

Flow cytometry:

• Quantification of GRIN2C expression levels in dissociated cells

• Enables high-throughput analysis of protein expression across cell populations

Colocalization studies:

• Investigation of GRIN2C spatial relationships with other proteins, such as 14-3-3 proteins that regulate receptor trafficking

• Research has shown important interactions between GRIN2C and regulatory proteins affecting receptor function and surface expression

Trafficking studies:

• Examination of GRIN2C surface expression and internalization dynamics

• Recent research has utilized FITC-conjugated antibodies to investigate how mutations affect GRIN2C trafficking and surface/total expression ratios

Each application requires specific optimization of fixation conditions, antibody concentration, incubation parameters, and imaging settings to achieve optimal signal-to-noise ratios.

How can GRIN2C antibody, FITC conjugated be used to investigate NMDA receptor trafficking in neurological disorders?

GRIN2C antibody with FITC conjugation provides a powerful tool for investigating altered NMDA receptor trafficking in neurological disorders. A methodological approach includes:

Surface expression quantification:

• Perform non-permeabilized immunostaining on cultured neurons to label only surface GRIN2C

• Follow with permeabilization and total GRIN2C labeling using a different fluorophore

• Calculate surface/total ratio to quantify trafficking alterations

Time-course trafficking studies:

• Utilize pulse-chase experiments with FITC-conjugated antibodies to track receptor internalization rates

• Compare trafficking dynamics between wild-type and mutant GRIN2C, or between control and disease conditions

Colocalization with trafficking machinery:
Recent research has revealed that GRIN2C A1072V mutation (associated with late-onset Alzheimer's disease) shows reduced colocalization between GRIN2C and 14-3-3 proteins, correlating with increased surface expression and enhanced NMDAR currents . This methodological approach involved:

• Transfecting primary hippocampal neurons with EGFP-tagged wild-type or mutant GRIN2C

• Performing immunocytochemistry with antibodies against EGFP and 14-3-3 proteins

• Analyzing colocalization using Pearson's correlation coefficient and Mander's overlap coefficient

• Correlating trafficking alterations with functional changes through electrophysiological recordings

Integration with functional studies:

• Combine imaging with whole-cell patch-clamp recordings to correlate trafficking alterations with functional changes

• Induce NMDA currents using controlled application of NMDA (400 μM) while monitoring electrical responses

• Apply NMDAR subunit-specific antagonists (e.g., QNZ46 for GluN2C) to verify subunit specificity

This integrated approach has revealed that mutant forms of GRIN2C can show significantly altered trafficking dynamics, contributing to disease pathophysiology through dysregulated glutamatergic signaling.

What strategies should be employed to detect low abundance GRIN2C expression in non-neuronal tissues?

Detecting low abundance GRIN2C in non-neuronal tissues presents unique challenges requiring specialized approaches:

Sample enrichment techniques:

• Perform subcellular fractionation to concentrate membrane proteins where GRIN2C is localized

• Use immunoprecipitation to concentrate GRIN2C before detection

• Consider proximity ligation assay (PLA) for enhanced sensitivity when detecting protein-protein interactions involving GRIN2C

Signal amplification methods:

• Implement tyramide signal amplification (TSA) to enhance FITC signal by up to 100-fold

• Use photomultiplier tube (PMT) detectors with increased gain settings during confocal microscopy

• Consider quantum dots coupled to secondary antibodies that recognize the FITC-conjugated primary antibody for increased photostability and brightness

Optimized fixation and antigen retrieval:

• Test multiple fixation protocols (e.g., 4% PFA, methanol, or acetone) to determine optimal epitope preservation

• Implement heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0)

• Extend primary antibody incubation time to 48-72 hours at 4°C to enhance binding to low-abundance targets

Combined transcript and protein detection:
Recent research examining GRIN2C in non-neuronal tissues has utilized a multi-level approach:

• Initial screening with RT-qPCR to confirm transcript presence

• Follow-up with immunofluorescence using higher antibody concentrations (1:20-1:50 range)

• Validation with multiple antibodies targeting different epitopes of GRIN2C

Comparison across tissues:
Include positive control samples from tissues known to express GRIN2C at high levels (e.g., cerebellum) for comparative analysis . Western blotting with increased protein loading (50-100 μg) may be required for initial validation before proceeding to imaging techniques.

These approaches have been successfully applied to detect GRIN2C in unexpected locations, such as cancer vasculature, where it may play roles in angiogenesis .

How can mutations in GRIN2C be investigated using FITC-conjugated antibodies in relation to Alzheimer's disease?

Recent research has identified rare damaging variants in GRIN2C associated with Alzheimer's disease, creating important research opportunities. A comprehensive methodological approach using FITC-conjugated antibodies includes:

Comparative expression analysis:

• Perform immunofluorescence on brain tissue sections from patients with GRIN2C mutations versus controls

• Quantify expression levels through standardized fluorescence intensity measurements

• Analyze regional distribution patterns across different brain areas affected in Alzheimer's disease

Trafficking alterations assessment:
A recent study investigating the GRIN2C A1072V variant in Alzheimer's disease used FITC-tagged antibodies to reveal:

• Increased surface/total ratio of mutant GRIN2C

• Reduced colocalization between GRIN2C and 14-3-3 regulatory proteins

• These alterations correlated with enhanced NMDAR-induced currents

The methodology involved:

• Transfecting primary hippocampal neurons with wild-type or mutant GRIN2C

• Using antibodies against an EGFP tag fused to GRIN2C to distinguish overexpressed protein from endogenous

• Performing surface labeling followed by permeabilization and total protein labeling

• Calculating surface/total ratios to quantify trafficking changes

Functional correlation:

• Combine imaging with electrophysiological recordings to assess how mutations affect receptor function

• Apply NMDA (400 μM) to induce currents while recording in whole-cell configuration

• Compare response magnitude between wild-type and mutant GRIN2C-expressing neurons

• Use GluN2C-specific antagonist QNZ46 (10 mM) to confirm subunit specificity of observed effects

Protein-protein interaction assessment:

• Perform co-immunoprecipitation followed by Western blotting to identify altered binding partners

• Use proximity ligation assay (PLA) for in situ detection of changed protein interactions

• Implement FRET (Fluorescence Resonance Energy Transfer) analysis between FITC-labeled GRIN2C and other fluorescently labeled interaction partners

This integrated approach has revealed that GRIN2C mutations can alter glutamatergic signaling through multiple mechanisms, potentially contributing to Alzheimer's disease pathogenesis through excitotoxicity or disrupted synaptic plasticity.

What are the optimal tissue preparation protocols for GRIN2C immunofluorescence with FITC-conjugated antibodies?

Optimal tissue preparation is critical for successful GRIN2C detection using FITC-conjugated antibodies. Based on recent experimental protocols, the following comprehensive approach is recommended:

Fixation optimization:

• Fresh tissue fixation: Immerse tissue in 4% paraformaldehyde (PFA) in PBS for 12-24 hours at 4°C

• Cryoprotection: Sequential sucrose gradients are essential - 20% sucrose for 3 days followed by 40% sucrose for 1 week

• Embedding: Use OCT compound and snap-freeze in isopentane cooled with dry ice

• Sectioning: Cut 20 μm sections using a cryostat and mount on positively charged slides

Antigen retrieval methods:
For GRIN2C detection, heat-mediated antigen retrieval often improves signal:

• Sodium citrate buffer (10 mM, pH 6.0) heating for 10 minutes at 95°C

• Allow slides to cool to room temperature for 20 minutes

• Wash thoroughly in PBS (3 × 5 minutes)

Permeabilization optimization:

• Use 0.1% Triton X-100 in PBS for 15 minutes at room temperature

• For dense tissues, consider increasing Triton X-100 concentration to 0.3%

Blocking parameters:

• Block with 5% bovine serum albumin (BSA) in PBS for 45-60 minutes at room temperature

• Include 5-10% normal serum from the same species as the tissue to reduce non-specific binding

Primary antibody application:

• Dilute FITC-conjugated GRIN2C antibody in blocking solution (typically 1:50-1:200)

• Incubate overnight at 4°C in a humid chamber protected from light

• For thick sections, consider extending incubation to 48-72 hours at 4°C

Nuclear counterstaining:
Use DAPI (1 μg/ml) for 5-10 minutes for nuclear visualization, avoiding spectral overlap with FITC.

Mounting considerations:

• Mount with an anti-fade mounting medium containing glycerol and n-propyl gallate

• For long-term storage, commercial mounting media containing anti-fade agents like ProLong Gold or Fluoroshield are recommended

These protocols have been successfully applied in research demonstrating GRIN2C expression in neural tissues and investigating its altered distribution in disease models.

How should FITC-conjugated GRIN2C antibodies be optimally stored and handled to preserve activity?

Proper storage and handling of FITC-conjugated GRIN2C antibodies is essential for maintaining their activity and fluorescence properties. Based on manufacturer recommendations and research practices, the following comprehensive protocol is advised:

Storage temperature:

• Store stock antibody solutions at -20°C in a non-frost-free freezer

• Stability is typically maintained for 12 months under these conditions

• Avoid storing at 4°C for extended periods as this accelerates degradation

Aliquoting strategy:

• Upon receipt, divide antibody into small single-use aliquots (10-20 μl)

• Use sterile microcentrifuge tubes with secure seals

• This practice minimizes freeze-thaw cycles and reduces contamination risk

Light protection measures:
FITC is particularly susceptible to photobleaching:

• Store in amber or opaque tubes, or wrap tubes in aluminum foil

• During experimental procedures, minimize exposure to light sources

• During microscopy, use minimum necessary exposure settings

Buffer composition:
Typical storage buffer contains:

• Phosphate buffered saline (PBS, pH 7.4)

• 0.02-0.05% sodium azide as preservative

• 50% glycerol as cryoprotectant
  Avoid altering this composition unless necessary.

Freeze-thaw management:

• Limit freeze-thaw cycles to maximum of 3-5

• Thaw antibodies slowly on ice, never at room temperature or by heating

• After thawing, mix gently by finger-flicking, not vortexing (which can denature antibodies)

Working dilution handling:

• Prepare working dilutions immediately before use

• If temporary storage is necessary, keep at 4°C for maximum 24-48 hours

• Protect diluted antibody from light and contamination

Quality monitoring:

• Include a positive control in each experiment to confirm antibody activity

• Monitor for signal deterioration over time, which indicates degradation

• Record lot numbers and correlate with performance to identify variation

Shipping and receiving:

• Upon receipt, immediately transfer to -20°C storage

• If shipping is necessary, use dry ice and express delivery

• Include temperature monitoring if shipping valuable antibody stocks

Following these guidelines will maximize the lifespan and consistent performance of FITC-conjugated GRIN2C antibodies, ensuring reliable experimental results.

What controls are essential for validating GRIN2C antibody specificity in immunofluorescence experiments?

Rigorous validation of GRIN2C antibody specificity requires a comprehensive set of controls to ensure accurate interpretation of experimental results:

Positive tissue controls:

• Include cerebellum sections where GRIN2C is known to be highly expressed

• Western blot validation using cerebellum lysate showing specific immunolabeling of the ~140 kDa GRIN2C protein

• Multiple brain regions with different GRIN2C expression levels for comparison

Negative tissue controls:

• Tissues known to lack GRIN2C expression

• Embryonic tissues before GRIN2C expression is established

• Cell lines that do not express GRIN2C naturally

Genetic validation controls:

• GRIN2C knockout or knockdown tissues/cells

• Heterozygous samples for dose-dependent validation

• Overexpression systems with tagged GRIN2C for positive control

Peptide competition assays:
Recent validation studies have shown that preincubation of the GRIN2C antibody with the specific immunogen peptide blocks labeling in Western blot and immunostaining applications . The protocol involves:

• Preincubating the antibody with 5-10 fold excess of immunizing peptide

• Parallel staining with blocked and unblocked antibody

• Complete abolishment of signal confirms specificity

Isotype controls:

• Use non-specific rabbit IgG at the same concentration as the GRIN2C antibody

• Apply identical staining protocol

• Assess background and non-specific binding

Antibody dilution series:

• Test multiple dilutions (e.g., 1:20, 1:50, 1:100, 1:200)

• Determine optimal signal-to-noise ratio

• Validate that staining pattern remains consistent across reasonable dilution range

Multi-method validation:

• Correlate immunofluorescence results with other detection methods:

  • In situ hybridization for GRIN2C mRNA

  • Western blot for protein expression

  • qPCR for transcript levels in the same tissue

Secondary antibody controls:

• Omit primary antibody to assess non-specific binding of detection systems

• For directly conjugated antibodies like FITC-GRIN2C, assess autofluorescence in unstained samples

Implementing these controls ensures that the observed staining truly represents GRIN2C distribution and avoids misinterpretation of experimental results.

What are the most effective approaches to reduce autofluorescence when using GRIN2C-FITC antibodies in brain tissue?

Brain tissue presents particular challenges for FITC-based immunofluorescence due to high autofluorescence. The following methodological approaches have proven effective:

Pre-treatment methods:

• Sodium borohydride treatment:

  • Incubate sections in freshly prepared 0.1% NaBH₄ in PBS for 10 minutes

  • Wash thoroughly in PBS (5 × 5 minutes)

  • Particularly effective for reducing aldehyde-induced autofluorescence

• Copper sulfate method:

  • Incubate sections in 1 mM CuSO₄ in 50 mM ammonium acetate buffer (pH 5.0) for 10-15 minutes

  • Effective for reducing lipofuscin autofluorescence common in aged brain tissue

• Sudan Black B treatment:

  • After immunostaining, incubate sections in 0.1-0.3% Sudan Black B in 70% ethanol for 10 minutes

  • Wash thoroughly with PBS containing 0.02% Tween-20

  • Particularly effective for reducing lipofuscin-related autofluorescence

Perfusion and fixation optimization:

• Perfuse animals with ice-cold PBS before fixative to clear blood (hemoglobin is highly autofluorescent)

• Minimize fixation time (12-24 hours optimal for most applications)

• Consider using lower concentrations of paraformaldehyde (2% instead of 4%) if autofluorescence persists

Spectral unmixing strategies:

• Acquire spectral images across multiple wavelengths

• Process with linear unmixing algorithms to separate FITC signal from autofluorescence

• Particularly useful for confocal microscopy with spectral detectors

Imaging parameters optimization:

• Use narrow bandpass filters centered precisely on FITC emission peak (515 nm)

• Implement time-gated imaging to exploit the different fluorescence lifetimes of FITC versus autofluorescence

• Consider using alternative conjugates (e.g., Alexa Fluor 488) which have higher quantum yield and photostability than FITC

Post-acquisition processing:

• Subtract background using unstained control sections

• Implement rolling ball background subtraction algorithms

• Use deconvolution to improve signal-to-noise ratio

These approaches have been successfully applied in research examining GRIN2C distribution in brain tissue, allowing for clear visualization even in challenging samples with high inherent autofluorescence.

How can FITC-conjugated GRIN2C antibodies be used in quantitative colocalization studies with other synaptic proteins?

Quantitative colocalization analysis requires meticulous experimental design and sophisticated analytical approaches. The following methodology optimizes FITC-conjugated GRIN2C antibody use in colocalization studies:

Sample preparation optimization:

• Use thin sections (10-15 μm) or cultured neurons for optimal resolution

• Implement identical fixation and permeabilization for all samples to ensure comparable protein retention

• Process all experimental groups simultaneously to minimize technical variation

Antibody selection and validation:

• Combine FITC-conjugated GRIN2C antibody with antibodies against synaptic markers (e.g., PSD-95, synapsin) conjugated to spectrally distinct fluorophores

• Validate specificity of each antibody individually before performing colocalization experiments

• Ensure fluorophore pairs have minimal spectral overlap (e.g., FITC and Alexa Fluor 647)

Image acquisition parameters:

• Use confocal microscopy with appropriate pinhole settings (0.5-1 Airy units)

• Acquire sequential scans rather than simultaneous to prevent cross-talk

• Establish standardized acquisition settings (laser power, gain, offset) and maintain across all samples

• Collect Z-stacks with Nyquist sampling to enable 3D colocalization analysis

• Include calibration samples with known degrees of colocalization

Quantitative analysis approaches:
Recent research investigating GRIN2C colocalization with regulatory proteins utilized:

• Pearson's correlation coefficient (PCC): Measures linear correlation between fluorescence intensities

• Mander's overlap coefficient (MOC): Quantifies fractional overlap, especially useful for proteins with different expression levels

• Intensity correlation analysis (ICA): Evaluates whether intensities of two channels vary in synchrony

Advanced analytical strategies:

• Implement object-based colocalization analysis:

  • Segment individual puncta using appropriate thresholding

  • Measure center-to-center distances between GRIN2C and synaptic protein puncta

  • Define colocalization based on distance criteria (typically <200 nm)

• Apply density-based colocalization:

  • Create density maps of protein distributions

  • Calculate correlation between density profiles

Controls for quantitative colocalization:

• Positive controls: Known interacting proteins that should show high colocalization

• Negative controls: Proteins known not to associate with GRIN2C

• Randomization controls: Create artificially randomized images to establish baseline "chance" colocalization

A recent study utilizing this approach revealed reduced colocalization between mutant GRIN2C (A1072V) and 14-3-3 proteins, correlating with altered surface expression and enhanced NMDAR currents in an Alzheimer's disease model .

What approaches can be used to study GRIN2C involvement in non-neuronal contexts such as cancer angiogenesis?

Recent research has identified novel roles for NMDA receptor subunits including GRIN2C in non-neuronal tissues, particularly in cancer-related angiogenesis . The following methodological approaches enable effective study of GRIN2C in these contexts:

Comparative expression analysis:

• Perform immunofluorescence with FITC-conjugated GRIN2C antibody on patient-matched tumor and normal tissues

• Quantify expression differences using standardized fluorescence intensity measurements

• Correlate GRIN2C expression with clinical outcomes and tumor stage

Cell type-specific localization:
Recent studies have identified NMDA receptor subunits in tumor vasculature . To investigate this:

• Perform multi-label immunofluorescence combining FITC-GRIN2C antibody with endothelial markers (CD31, CD34)

• Use confocal microscopy to determine precise cellular localization

• Quantify colocalization using Pearson's correlation coefficient or Mander's overlap coefficient

Functional validation approaches:

• siRNA knockdown studies:

  • Design and validate siRNAs targeting GRIN2C

  • Transfect endothelial cells or cancer cells and confirm knockdown by qPCR and immunofluorescence

  • Assess effects on angiogenic functions (migration, tube formation, proliferation)

• Overexpression studies:

  • Generate GRIN2C overexpression constructs with fluorescent tags

  • Transfect cells and confirm expression by immunofluorescence

  • Examine effects on angiogenic phenotypes

• Pharmacological manipulation:

  • Apply GluN2C-specific modulators (e.g., CIQ as a positive allosteric modulator)

  • Assess effects on angiogenic processes and signaling pathways

  • Combine with immunofluorescence to correlate receptor distribution with functional outcomes

In vivo angiogenesis models:
Research has demonstrated that targeting NMDA receptor subunits can inhibit tumor angiogenesis . To investigate this:

• Implement subcutaneous sponge angiogenesis assays in combination with GRIN2C targeting approaches

• Utilize tumor xenograft models to assess effects of GRIN2C modulation on tumor vascularization

• Perform immunofluorescence on harvested tissues to correlate intervention with GRIN2C expression

Mechanistic investigations:

• Examine calcium signaling in endothelial cells expressing GRIN2C using calcium imaging techniques

• Investigate downstream signaling pathways activated by GRIN2C in endothelial cells

• Identify binding partners specific to non-neuronal GRIN2C function through co-immunoprecipitation followed by mass spectrometry