GSX2 Antibody, FITC conjugated

What is the target specificity of GSX2 Antibody, FITC conjugated?

GSX2 Antibody, FITC conjugated specifically targets the amino acid sequence 15-118 of the human GSX2 protein (also known as GSH2, Genetic-screened homeobox 2, or Homeobox protein GSH-2) . This region is part of the N-terminal domain of the protein, which is distinct from the DNA-binding homeodomain. The antibody is generated using recombinant human GSX2 protein fragment as the immunogen and demonstrates high specificity for human samples . When designing experiments, researchers should consider that while the antibody has been validated for human samples, cross-reactivity with other mammalian species may occur due to sequence conservation but should be empirically validated before proceeding with non-human models .

What are the recommended storage conditions for preserving FITC-conjugated GSX2 antibody activity?

For optimal preservation of fluorescent signal and antibody activity, GSX2 Antibody, FITC conjugated should be stored at -20°C or -80°C upon receipt . The antibody is provided in a liquid format with a specialized buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 . This formulation helps maintain antibody stability and prevent microbial growth. Repeated freeze-thaw cycles should be avoided as they can degrade both the antibody protein and the FITC fluorophore, resulting in decreased signal intensity and increased background . Working aliquots should be prepared upon first thaw to minimize repeated exposure to room temperature, and the antibody should be protected from light during handling to prevent photobleaching of the FITC fluorophore.

What experimental applications is the GSX2 Antibody, FITC conjugated suitable for?

While specific applications for the FITC-conjugated version may require further inquiry, GSX2 antibodies generally demonstrate utility in multiple experimental techniques . The FITC conjugation makes this antibody particularly suitable for direct immunofluorescence applications such as flow cytometry (FACS), fluorescence microscopy, and immunohistochemistry where direct visualization is advantageous. For the specific FITC-conjugated antibody targeting AA 15-118, ELISA applications have been validated . When designing experiments, researchers should consider additional validation for other intended applications, as conjugation can sometimes affect binding characteristics compared to unconjugated versions of the same antibody. Preliminary titration experiments to determine optimal working concentrations are recommended before proceeding with full-scale studies.

How can GSX2 Antibody, FITC conjugated be optimized for neural stem cell research applications?

Given GSX2's critical role in neural stem cell (NSC) activation and olfactory bulb interneuron generation , researchers studying NSC populations should implement specific optimization strategies. When performing immunofluorescence on neural tissues, antigen retrieval methods should be carefully selected as GSX2 is a nuclear transcription factor that may require enhanced nuclear permeabilization. For fixed tissue sections, a combination of heat-mediated antigen retrieval in citrate buffer (pH 6.0) followed by permeabilization with 0.3% Triton X-100 has shown effectiveness for nuclear transcription factors.

For flow cytometry applications studying NSCs, researchers should implement a sequential staining protocol: first using surface markers (CD133, Nestin) to identify NSC populations, followed by fixation, permeabilization, and nuclear staining with the GSX2-FITC antibody. This approach allows for correlation between GSX2 expression levels and specific NSC subpopulations. Additionally, when co-staining with other fluorophores, researchers should carefully select compatible fluorophores that have minimal spectral overlap with FITC (excitation ~495nm, emission ~520nm) to reduce compensation requirements and improve signal discrimination.

What methodological approaches can address potential autofluorescence interference when using GSX2 Antibody, FITC conjugated in brain tissue?

When working with brain tissue samples, autofluorescence presents a significant challenge for FITC-conjugated antibodies due to lipofuscin accumulation, particularly in aged specimens. To mitigate this interference, researchers should implement a comprehensive autofluorescence reduction strategy. Prior to immunostaining, treat sections with 0.1% Sudan Black B in 70% ethanol for 20 minutes, followed by thorough washing. This treatment significantly reduces lipofuscin-derived autofluorescence while preserving specific FITC signals.

For confocal microscopy applications, employ sequential scanning and narrow bandpass filters to better discriminate between specific GSX2-FITC signals and broad-spectrum autofluorescence. Additionally, implement spectral unmixing algorithms during image analysis to computationally separate FITC signals from autofluorescence patterns. When studying GSX2 in regions known for high autofluorescence (such as substantia nigra), consider using alternative conjugates like Cy5 that operate in spectral ranges with lower endogenous fluorescence. Always include appropriate negative controls (secondary-only for indirect methods, or isotype controls for directly conjugated antibodies) to establish baseline autofluorescence levels for each experimental condition.

How can GSX2 Antibody, FITC conjugated be utilized for quantitative assessment of GSX2 expression during neuronal differentiation?

For quantitative tracking of GSX2 expression during neural differentiation, a multiparametric approach is essential. Flow cytometry offers the most rigorous quantitative assessment, where median fluorescence intensity (MFI) values can be measured at different time points during differentiation protocols. Establish a calibration curve using quantitative fluorescence calibration beads to convert arbitrary fluorescence units to molecules of equivalent soluble fluorochrome (MESF), enabling standardized measurements across experiments and instruments.

For in situ quantification in adherent cultures or tissue sections, implement automated image analysis workflows using open-source platforms like CellProfiler or commercial solutions like Imaris. Define nuclear masks based on DAPI staining, then measure integrated FITC intensity within these nuclear regions. This approach allows population-level analysis of GSX2 expression changes. For developmental studies tracking GSX2 during telencephalic development, time-course experiments should include co-staining with markers of differentiation states (Nestin, Tuj1, MAP2) to correlate GSX2 levels with specific developmental transitions. Statistical analysis should employ mixed-effects models to account for both biological and technical variability in longitudinal studies.

What controls are essential when using GSX2 Antibody, FITC conjugated for immunofluorescence studies?

Implementing comprehensive controls is critical for accurate interpretation of GSX2-FITC immunofluorescence data. At minimum, researchers should include:

• Positive control: Transfected cell lysates overexpressing GSX2 protein or tissues known to express high levels of GSX2 (such as specific regions of embryonic brain) . This confirms antibody functionality under your experimental conditions.

• Negative control: Samples where GSX2 expression is absent or knocked down (via siRNA or CRISPR). For tissue sections, regions known to lack GSX2 expression can serve as internal negative controls.

• Isotype control: A FITC-conjugated rabbit IgG targeting an irrelevant antigen, used at the same concentration as the GSX2 antibody, to assess non-specific binding of the antibody scaffold.

• Absorption control: Pre-incubate the GSX2-FITC antibody with excess recombinant GSX2 protein (15-118AA) before staining to demonstrate binding specificity.

• Single-color controls: When performing multicolor experiments, include single-color controls for each fluorophore to establish compensation settings and assess spectral overlap.

These controls help distinguish between specific GSX2 staining and artifacts arising from autofluorescence, non-specific binding, or technical issues with the staining protocol.

How can discrepancies between GSX2 protein detection methods be reconciled in research applications?

When researchers encounter discrepancies between different detection methods for GSX2 (e.g., immunofluorescence showing different results than Western blot), a systematic troubleshooting approach is necessary. First, consider epitope accessibility: the FITC-conjugated antibody targets amino acids 15-118 , while other antibodies may target different regions such as the C-terminus (AA 243-269) . Post-translational modifications or protein-protein interactions may differentially mask these epitopes across experimental conditions.

For quantitative comparisons between methods, normalize GSX2 measurements to appropriate housekeeping controls for each technique (GAPDH for Western blots, nuclear area measurements for immunofluorescence). When significant discrepancies persist, employ orthogonal validation through mRNA quantification (qPCR) or mass spectrometry. Consider that antibody performance can vary between batches; therefore, maintain detailed records of lot numbers and validation experiments. For definitive resolution of conflicting results, combine multiple antibodies targeting different GSX2 epitopes within the same experiment, or validate findings using genetic approaches (overexpression or knockdown) to confirm the specificity of observed signals.

What is the recommended protocol for dual immunofluorescence labeling when combining GSX2 Antibody, FITC conjugated with other neural markers?

When performing dual immunofluorescence to co-localize GSX2 with other neural markers, the direct FITC conjugation requires specific protocol adaptations. The following sequential staining approach is recommended:

• Fixation and permeabilization: Fix samples with 4% paraformaldehyde for 15 minutes, followed by permeabilization with 0.3% Triton X-100 in PBS for 10 minutes (critical for nuclear transcription factor access).

• Blocking: Incubate with 5% normal serum (species different from primary antibodies) containing 0.1% Triton X-100 for 1 hour at room temperature.

• Primary antibody for second marker: Apply unconjugated primary antibody against the complementary neural marker (e.g., anti-Nestin, anti-DLX2) overnight at 4°C.

• Secondary antibody: Apply species-specific secondary antibody conjugated to a fluorophore with minimal spectral overlap with FITC (e.g., Cy3, Alexa 594, or Alexa 647) for 1 hour at room temperature.

• GSX2-FITC application: After washing, apply the GSX2-FITC conjugated antibody at optimized concentration for 2 hours at room temperature or overnight at 4°C.

• Nuclear counterstain: Apply DAPI (1:1000) for 5 minutes to visualize nuclei.

• Mounting: Mount using anti-fade mounting medium to preserve FITC fluorescence.

This sequential approach minimizes cross-reactivity and optimizes signal-to-noise ratio for both markers. For markers with weak expression, consider using tyramide signal amplification (TSA) systems for the non-FITC channel to balance signal intensities between the directly conjugated GSX2-FITC and the indirectly detected marker.

How can GSX2 Antibody, FITC conjugated be utilized to investigate the role of GSX2 in defining neuronal subtype specification during development?

Given GSX2's role in specifying different neuronal fates depending on developmental stage , the FITC-conjugated antibody can be implemented in a temporally controlled experimental design. For developmental studies, time-course experiments should be conducted across multiple embryonic stages (E12.5-E18.5 in mice) and early postnatal periods to capture the transition from early ventral lateral ganglionic eminence (LGE) specification to later roles in olfactory bulb interneuron development .

For each developmental timepoint, perform triple immunofluorescence using GSX2-FITC alongside stage-specific markers and region-specific markers. For example, co-stain with Dlx2 (a downstream target of GSX2) and Isl1 (a striatal projection neuron marker) at early stages, transitioning to Sp8 and CalR (olfactory interneuron markers) at later stages. Quantitative image analysis should track both the percentage of GSX2+ cells and the fluorescence intensity per nucleus across development, correlating these measurements with the emergence of different neuronal subtypes.

For in vitro applications using neural stem cell differentiation models, implement time-lapse imaging with the GSX2-FITC antibody in live cell-compatible fixation protocols to track temporal dynamics of GSX2 expression during the differentiation process. This approach allows for correlation between GSX2 expression patterns and subsequent neuronal subtype specification, providing insights into the molecular mechanisms governing fate decisions in the developing telencephalon.

What methodological considerations are essential when using GSX2 Antibody, FITC conjugated for quantitative comparison of expression levels across different brain regions?

When comparing GSX2 expression quantitatively across brain regions, several methodological considerations must be addressed to ensure valid comparisons. First, tissue processing must be standardized: all regions should undergo identical fixation protocols, as fixation duration and reagents can affect epitope accessibility and fluorescence intensity. Section thickness should be consistent (recommended 30-40μm for adult brain tissue), and sections should be processed in parallel batches.

For imaging, establish standardized acquisition parameters: use identical exposure settings, laser power, detector gain, and pinhole settings across all regions. Include fluorescence calibration standards in each imaging session to normalize for day-to-day variations in system performance. For regional comparisons, define anatomical regions using consistent neuroanatomical landmarks or stereotaxic coordinates, and analyze multiple sections spanning each region.

Data analysis should implement a multi-level approach:

• Cell counting: Determine the percentage of GSX2+ cells within defined regions

• Intensity measurement: Quantify mean and integrated GSX2-FITC intensity within individual nuclei

• Distribution analysis: Assess the population distribution of GSX2 expression levels using histogram or violin plots

To account for regional variations in autofluorescence, implement region-specific background subtraction based on measurements from control sections. Statistical analysis should employ nested ANOVA or linear mixed-effects models that account for the hierarchical nature of the data (multiple measurements within sections, multiple sections within animals, multiple animals within experimental groups).

How can GSX2 Antibody, FITC conjugated be adapted for multiparametric flow cytometry to isolate neural progenitor subpopulations?

For multiparametric flow cytometry applications isolating GSX2+ neural progenitor subpopulations, a comprehensive panel design is essential. Given GSX2's role in neural stem cell activation and specification , combine the GSX2-FITC antibody with surface and intracellular markers that define neural progenitor states and lineage progression:

Suggested 8-color panel for neural progenitor analysis:

• GSX2-FITC: Transcription factor of interest

• CD133-PE: Neural stem cell marker

• EGFR-PE-Cy7: Activated neural stem/progenitor marker

• PSA-NCAM-APC: Neuroblast marker

• DLX2-Alexa 647: Ventral telencephalic marker

• ASCL1-BV421: Proneural transcription factor

• Ki67-BV510: Proliferation marker

• DAPI: Viability/DNA content

Sample preparation requires careful optimization of fixation and permeabilization protocols that preserve both surface epitopes and nuclear transcription factors. A sequential staining approach is recommended:

• Isolate cells using gentle enzymatic digestion (papain-based neural dissociation)

• Stain for surface markers (CD133, EGFR, PSA-NCAM) on live cells

• Fix with 2% PFA for 15 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for nuclear factors

• Stain with intracellular/nuclear antibodies (GSX2-FITC, DLX2, ASCL1, Ki67)

For analysis, implement a hierarchical gating strategy that first identifies viable single cells (DAPI low, appropriate FSC/SSC), then progressively narrows to neural stem/progenitor populations before examining GSX2 expression within defined subpopulations. Use fluorescence-minus-one (FMO) controls to set accurate gates for GSX2 positivity, and include isotype controls to assess non-specific binding. This approach enables isolation of distinct GSX2+ progenitor subpopulations based on their developmental stage and regional identity, facilitating subsequent functional or molecular characterization through cell sorting.

How might GSX2 Antibody, FITC conjugated be utilized in combination with advanced imaging techniques to elucidate GSX2 dynamics in neural development?

The GSX2 Antibody, FITC conjugated can be integrated with cutting-edge imaging technologies to reveal previously uncharacterized aspects of GSX2 function in neural development. Super-resolution microscopy techniques such as Structured Illumination Microscopy (SIM) or Stochastic Optical Reconstruction Microscopy (STORM) can be applied to visualize the subnuclear distribution of GSX2 at nanoscale resolution, potentially revealing relationships between GSX2 localization and transcriptional activity that are not visible with conventional microscopy . These approaches might identify specific nuclear compartments or chromatin regions where GSX2 preferentially localizes during different developmental stages.

For in vivo applications, tissue clearing methodologies (CLARITY, iDISCO+, or CUBIC) can be combined with light-sheet microscopy to visualize GSX2 expression patterns throughout intact embryonic brains. This would provide comprehensive 3D mapping of GSX2+ progenitor domains and their relationship to emerging brain structures. Researchers could track the progeny of GSX2+ progenitors using genetic fate mapping approaches (such as GSX2-CreERT2 driver lines) in combination with clearing techniques and GSX2-FITC immunostaining to simultaneously visualize both current GSX2 expression and historically GSX2-derived populations.

These advanced imaging approaches, when combined with computational spatial analysis, would enable quantitative modeling of how GSX2 expression gradients relate to emerging brain regional specification and neuronal subtype diversity.

What considerations are important when designing experiments to investigate GSX2 protein-protein interactions using FITC-conjugated antibodies?

When investigating GSX2 protein-protein interactions using FITC-conjugated antibodies, researchers must carefully consider how the FITC moiety might affect detection of interaction partners. For co-immunoprecipitation experiments, the FITC conjugation may interfere with certain protein-protein interactions if the fluorophore is located near interaction domains within the 15-118 amino acid region of GSX2 . Alternative approaches include using the FITC-conjugated antibody for detection after immunoprecipitation with an unconjugated antibody targeting a different GSX2 epitope, such as a C-terminal region (AA 243-269) .

For in situ visualization of protein interactions, proximity ligation assays (PLA) offer high sensitivity. In this approach, the GSX2-FITC antibody would be paired with an unconjugated primary antibody against a suspected interaction partner, followed by secondary PLA probes that generate a fluorescent signal only when the two proteins are in close proximity (<40 nm). This approach produces discrete spots that can be quantified to assess interaction frequency and cellular localization.

Advanced fluorescence techniques such as Förster Resonance Energy Transfer (FRET) can also be employed when the FITC on the GSX2 antibody serves as a donor fluorophore, paired with an acceptor fluorophore (such as Cy3 or TRITC) on an antibody targeting a potential interaction partner. Successful energy transfer, measured as donor quenching or acceptor sensitization, indicates molecular proximity consistent with protein-protein interaction.

In all cases, appropriate controls are essential, including negative controls (proteins not expected to interact with GSX2) and positive controls (known GSX2 interaction partners) to validate the specificity and sensitivity of the chosen method.