KLF2 Antibody, FITC conjugated

What is KLF2 and why is it significant in biological research?

KLF2 (Krüppel-like factor 2) is a transcription factor that regulates diverse biological processes, including development, differentiation, proliferation, growth, survival, and responses to external stresses. It plays a crucial role in the differentiation and function of immune cells and vascular endothelial cells. KLF2 is particularly significant because it suppresses NF-κB-mediated gene expression and is important in inflammatory responses . Alterations in KLF2 function have been linked to various human diseases, including metabolic diseases and cancer, making it an important target for research . Recent studies have also demonstrated KLF2's renoprotective effects on glomerular endothelial cells, suggesting its potential role in hypertensive nephropathy .

What are the primary applications of FITC-conjugated KLF2 antibodies?

FITC-conjugated KLF2 antibodies are versatile tools primarily used in fluorescence-based detection techniques. While standard KLF2 antibodies can be used in Western Blotting (1:1000 dilution), Immunoprecipitation (1:50 dilution), Immunohistochemistry on paraffin-embedded samples (1:100-1:400), Immunofluorescence (1:800-1:1600), and Flow Cytometry (1:100-1:400) , FITC-conjugated versions are particularly valuable for flow cytometry and immunofluorescence applications. The FITC conjugation eliminates the need for secondary antibodies in fluorescence-based detection systems, simplifying experimental protocols and reducing background in multicolor analysis . These antibodies are essential for studying KLF2 expression in specific cell populations, tissue localization, and for investigating its role in disease models.

How does KLF2 function compare to other KLF family members?

KLF2 is one of seventeen mammalian KLF proteins identified to date, all of which are related to the Drosophila melanogaster Krüppel protein that regulates body segmentation . While all KLF family members share a DNA-binding domain consisting of three zinc fingers, KLF2 has unique regulatory roles. Unlike some other KLF members that promote cell proliferation, KLF2 generally inhibits cell proliferation and promotes quiescence in certain cell types. In the context of T cells, KLF2 restrains CD4+ T follicular helper cell differentiation, distinguishing it from KLF4, which has different roles in immune regulation . In endothelial cells, KLF2 has complementary but distinct functions from KLF4, as evidenced by studies showing γ-protocadherins can inhibit both KLF2 and KLF4 to influence vascular remodeling .

What is the optimal protocol for using FITC-conjugated KLF2 antibodies in flow cytometry?

For optimal flow cytometry results with FITC-conjugated KLF2 antibodies, the following protocol is recommended:

• Cell preparation: Harvest cells (1-5 × 10^6) and wash twice with PBS containing 1% BSA.

• Fixation: Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature.

• Permeabilization: Wash cells and permeabilize with 0.1% Triton X-100 in PBS for 5-10 minutes.

• Blocking: Incubate cells with blocking buffer (PBS containing 5% normal serum from the same species as the secondary antibody) for 30 minutes.

• Antibody staining: Add FITC-conjugated KLF2 antibody at a dilution of 1:100-1:400 and incubate for 30-60 minutes in the dark at room temperature .

• Washing: Wash cells three times with PBS containing 1% BSA.

• Analysis: Analyze on a flow cytometer with appropriate filters for FITC detection (excitation at 488 nm, emission at 530 nm).

For intracellular staining, ensure proper fixation and permeabilization as KLF2 is a nuclear transcription factor. The buffer should contain preservative (0.03% Proclin 300) and be stored in 50% glycerol and 0.01M PBS at pH 7.4 for optimal stability .

How should researchers optimize immunofluorescence protocols for KLF2 detection in tissue samples?

Optimizing immunofluorescence protocols for KLF2 detection requires attention to several key parameters:

• Tissue preparation:

  • For paraffin-embedded sections: Deparaffinize, rehydrate, and perform antigen retrieval (citrate buffer, pH 6.0, at 95-100°C for 20 minutes).

  • For frozen sections: Fix with 4% paraformaldehyde and permeabilize with 0.2% Triton X-100.

• Blocking: Block with 5-10% normal serum and 1% BSA in PBS for 1 hour at room temperature to reduce background.

• Primary antibody incubation: Apply FITC-conjugated KLF2 antibody at a dilution of 1:800-1:1600 . Incubate overnight at 4°C in a humidified chamber protected from light.

• Washing: Perform at least three 5-minute washes with PBS containing 0.05% Tween-20.

• Nuclear counterstaining: Use DAPI (1 μg/mL) for 5 minutes to visualize nuclei.

• Mounting: Mount with an anti-fade mounting medium to prevent photobleaching.

For double or triple immunofluorescence staining, select additional primary antibodies from different species and appropriate fluorophores with minimal spectral overlap with FITC. When studying glomerular endothelial cells, co-staining with endothelial markers like CD31 can help confirm the specificity of KLF2 localization, particularly important in hypertensive nephropathy models .

What controls should be included in experiments using FITC-conjugated KLF2 antibodies?

A robust experimental design using FITC-conjugated KLF2 antibodies should include the following controls:

• Isotype control: Use FITC-conjugated rabbit IgG (the same isotype as the KLF2 antibody) at the same concentration to assess non-specific binding .

• Negative controls:

  • Unstained cells/tissues to establish autofluorescence levels

  • Secondary antibody only (for protocols using unconjugated primary antibodies)

  • Cells known to have very low or no expression of KLF2

• Positive controls:

  • Cell lines or tissues with documented KLF2 expression

  • Recombinant KLF2 protein for Western blot applications

• Knockdown/knockout validation: Where possible, include KLF2 siRNA-treated samples to confirm antibody specificity, as demonstrated in studies examining pressure-induced fibrosis in glomerular endothelial cells .

• Blocking peptide control: Pre-incubation of the antibody with a blocking peptide (the immunogen) should abolish specific staining.

For flow cytometry experiments, include single-color controls for compensation when performing multicolor analyses, and use fluorescence-minus-one (FMO) controls to determine gating boundaries accurately.

How can researchers effectively use KLF2 antibodies to study endothelial dysfunction in hypertension models?

To effectively study endothelial dysfunction in hypertension using KLF2 antibodies, researchers should implement a multi-faceted approach:

• In vitro hypertension modeling: Utilize specialized equipment, such as pressure-application devices that can apply controlled pressure (4-8 mmHg) to human primary glomerular endothelial cells (hPGECs) to mimic hypertensive conditions . After pressure application, assess KLF2 expression levels using immunofluorescence or flow cytometry with FITC-conjugated KLF2 antibodies.

• Fibrotic marker correlation: Measure the expression of fibrotic markers such as fibronectin and TGF-β in relation to KLF2 levels. Studies have shown that pressure-induced fibrosis increases fibronectin-positive cells from 25.2% under static conditions to 59.6% under 4 mmHg pressure and 69.2% under 8 mmHg pressure .

• Manipulating KLF2 expression:

  • Upregulate KLF2 using statins (e.g., simvastatin at 10 μM), which has been shown to decrease pressure-induced fibrosis to 45.7% fibronectin-positive cells

  • Downregulate KLF2 using siRNA transfection, which increases fibrosis markers to 71.4% fibronectin-positive cells

• Animal model validation: Utilize 5/6 nephrectomy mouse models to study hypertensive nephropathy, and use FITC-conjugated KLF2 antibodies for immunohistochemistry to assess glomerular KLF2 expression patterns .

• Human sample correlation: Compare findings with kidney biopsy samples from patients with hypertensive nephropathy, examining KLF2 expression in glomerular endothelial cells to establish clinical relevance.

This comprehensive approach allows for mechanistic understanding of how KLF2 regulates endothelial responses to hypertension and may identify potential therapeutic targets.

What are the technical challenges in studying KLF2 localization in T cell subpopulations?

Studying KLF2 localization in T cell subpopulations presents several technical challenges:

• Dynamic expression patterns: KLF2 expression fluctuates during T cell differentiation and activation, making timing of analysis critical. Research has shown that KLF2 restrains CD4+ T follicular helper cell differentiation, and its deletion increases the frequency and number of Tfh phenotype CD4+ T cells .

• Nuclear localization: As a transcription factor, KLF2 is predominantly nuclear, requiring effective cell permeabilization protocols that maintain cell surface markers for subset identification.

• Low abundance: Transcription factors are often expressed at relatively low levels, necessitating signal amplification strategies or highly sensitive detection methods.

• T cell subset identification: Simultaneous staining for multiple lineage markers requires careful panel design to avoid spectral overlap with FITC. Consider the following multicolor panel:

  • FITC-conjugated KLF2 antibody

  • PE-conjugated anti-CD4

  • APC-conjugated anti-CXCR5 (for Tfh identification)

  • PE-Cy7-conjugated anti-PD-1

  • BV421-conjugated anti-CD44 (activation marker)

• Fixation-sensitive epitopes: Some T cell surface markers may be sensitive to fixation/permeabilization, requiring protocol optimization or alternative marker selection.

• Ex vivo versus in vitro analysis: T cells rapidly alter their phenotype ex vivo, potentially affecting KLF2 expression. Consider using in situ imaging of lymphoid tissues when possible.

To address these challenges, consider using imaging flow cytometry, which combines the quantitative power of flow cytometry with the visual confirmation of imaging, allowing for accurate assessment of KLF2 nuclear localization in identified T cell subsets.

How can KLF2 antibodies be utilized in investigating pressure-induced endothelial cell apoptosis and fibrosis mechanisms?

KLF2 antibodies are invaluable tools for investigating pressure-induced endothelial cell apoptosis and fibrosis through several methodological approaches:

• Pressure-application models: Utilize specialized devices that apply rotational force to cultured human primary glomerular endothelial cells (hPGECs) to simulate hypertensive conditions. Apply varying pressures (static, 4 mmHg, 8 mmHg) and assess KLF2 expression using immunofluorescence with FITC-conjugated antibodies .

• Dual immunofluorescence staining: Combine FITC-conjugated KLF2 antibodies with markers for:

  • Apoptosis (PE-conjugated Annexin V or antibodies against cleaved caspase-3)

  • Fibrosis (antibodies against fibronectin, α-SMA, and TGF-β)

  • Angiotensin II and its receptors (AT1R, AT2R)

  • Inflammatory markers (IL-18)

• KLF2 manipulation strategies:

  • Pharmacological upregulation using simvastatin (10 μM)

  • Genetic downregulation using KLF2 siRNA (2 μM)

  • Monitor resulting changes in apoptosis and fibrosis markers

• Quantitative analysis protocols:

  • Flow cytometry to quantify the proportion of fibronectin-positive cells (which increases from 25.2% under static conditions to 59.6% under 4 mmHg pressure)

  • qRT-PCR to measure mRNA expression of TGF-β, fibronectin, angiotensin II, and its receptors

  • Western blotting to assess protein levels of apoptotic and fibrotic markers

• Mechanistic pathway elucidation: Use FITC-conjugated KLF2 antibodies in combination with inhibitors of specific signaling pathways (AT1R blockers, TGF-β inhibitors) to delineate the molecular mechanisms by which KLF2 regulates pressure-induced endothelial damage.

This comprehensive approach can reveal how pressure-induced apoptosis and fibrosis of glomerular endothelial cells result from angiotensin II, AT1R activation, and KLF2 inhibition, potentially mediated by inflammatory factors like IL-18 .

How should researchers interpret contradictory results between KLF2 antibody detection and mRNA expression data?

When faced with discrepancies between KLF2 protein detection using antibodies and mRNA expression data, consider the following interpretative framework:

• Post-transcriptional regulation: KLF2 is subject to extensive post-transcriptional regulation, including microRNA-mediated repression and protein stability control. Verify if the discrepancy might reflect biological regulation rather than technical artifacts.

• Technical considerations:

  • Antibody specificity: Confirm the KLF2 antibody detects the correct molecular weight (42 kDa) and shows reduced signal in KLF2 knockdown experiments.

  • mRNA detection methods: qRT-PCR primers might detect specific splice variants not recognized by the antibody, or vice versa.

  • Tissue/cell heterogeneity: In mixed cell populations, determine if different cell types contribute disproportionately to mRNA versus protein signals.

• Temporal dynamics: KLF2 mRNA and protein may have different half-lives and expression kinetics. Time-course experiments can help resolve temporal discrepancies.

• Cross-validation approaches:

  • Use multiple antibodies targeting different epitopes of KLF2

  • Employ alternative mRNA detection methods (RNA-seq, Northern blot)

  • Perform in situ hybridization alongside immunofluorescence on the same samples

• Functional validation: Perform functional assays (e.g., reporter assays for KLF2 transcriptional activity) to determine which measurement better correlates with biological function.

In studies of pressure-induced endothelial damage, researchers observed coordinated changes in both KLF2 mRNA and protein levels, providing a useful reference for expected concordance under certain experimental conditions .

What are the most common sources of false positives/negatives when using FITC-conjugated KLF2 antibodies and how can they be mitigated?

Common sources of false results with FITC-conjugated KLF2 antibodies and mitigation strategies include:

Sources of False Positives:

• Autofluorescence: Cellular components, particularly in tissues rich in elastin, collagen, or lipofuscin, can generate green autofluorescence in the FITC spectrum.

  • Mitigation: Include unstained controls and use spectral unmixing or autofluorescence quenching reagents.

• Non-specific binding: The antibody may bind to Fc receptors or proteins with similar epitopes.

  • Mitigation: Use appropriate blocking with serum (5-10%) and include isotype controls (FITC-conjugated rabbit IgG) .

• Cross-reactivity: The antibody may detect related KLF family members.

  • Mitigation: Verify specificity using KLF2-knockout or knockdown samples, and check the datasheet for cross-reactivity testing .

• Fluorophore transfer: FITC can sometimes dissociate from antibodies and bind non-specifically.

  • Mitigation: Use fresh antibody preparations and proper storage (avoid repeated freeze-thaw cycles) .

Sources of False Negatives:

• Epitope masking: Fixation may alter KLF2 epitopes, particularly in formalin-fixed tissues.

  • Mitigation: Optimize antigen retrieval methods (citrate buffer, pH 6.0) and test multiple fixation protocols.

• Insufficient permeabilization: As a nuclear transcription factor, KLF2 requires adequate nuclear access.

  • Mitigation: Optimize permeabilization with different detergent concentrations and incubation times.

• Fluorophore quenching: FITC is susceptible to photobleaching.

  • Mitigation: Minimize light exposure during staining, use anti-fade mounting media, and acquire images promptly.

• Protein degradation: KLF2 may be labile in certain sample preparation conditions.

  • Mitigation: Use fresh samples when possible and include protease inhibitors during preparation.

• Insufficient sensitivity: KLF2 expression may be below detection limits in some cell types.

  • Mitigation: Consider signal amplification methods or more sensitive detection systems.

Always validate findings with complementary techniques (Western blotting, qRT-PCR) and include appropriate positive controls, such as tissues known to express KLF2 (e.g., lung endothelial cells).

How can researchers accurately quantify KLF2 expression levels across different experimental conditions?

Accurate quantification of KLF2 expression across experimental conditions requires a structured approach:

• Flow cytometry quantification:

  • Use median fluorescence intensity (MFI) rather than percent positive cells for more precise comparisons

  • Include calibration beads with known fluorophore numbers to convert MFI to absolute molecule numbers

  • Apply consistent gating strategies across all samples

  • Report fold-change relative to control conditions

  Example data table for flow cytometry quantification:

  Experimental Condition	KLF2 MFI	Fold Change vs Control	% Positive Cells
  Static (Control)	345 ± 28	1.0	25.2 ± 3.6%
  4 mmHg Pressure	195 ± 17	0.57	59.6 ± 5.6%
  8 mmHg Pressure	158 ± 21	0.46	69.2 ± 3.0%
  4 mmHg + Simvastatin	312 ± 30	0.90	45.7 ± 5.7%
  4 mmHg + KLF2 siRNA	98 ± 15	0.28	71.4 ± 22.9%

• Immunofluorescence quantification:

  • Use digital image analysis software to quantify nuclear FITC intensity

  • Apply threshold-based segmentation to identify nuclei (using DAPI)

  • Measure integrated optical density or mean pixel intensity of KLF2 staining within nuclear regions

  • Analyze at least 100-200 cells per condition across multiple fields

• Western blotting standardization:

  • Use recombinant KLF2 protein standards to generate a calibration curve

  • Normalize KLF2 signals to appropriate loading controls (e.g., nuclear proteins like Lamin B for nuclear extracts)

  • Apply the 1:1000 dilution recommended for the antibody

  • Quantify band intensity using densitometry software

• mRNA expression analysis:

  • Use absolute quantification with standard curves when possible

  • Select stable reference genes validated for your experimental conditions

  • Apply the 2^-ΔΔCt method for relative quantification

  • Include no-template and no-RT controls

• Multi-method integration:

  • When possible, correlate protein levels (by immunofluorescence or Western blotting) with mRNA expression

  • Use statistical approaches like Pearson correlation to assess the relationship between different quantification methods

  • Report concordant and discordant results transparently

When reporting quantification results, include both raw values and normalized/relative values, clearly state the normalization method, and provide measures of statistical variability (standard deviation or standard error) from multiple biological replicates.

What emerging techniques might enhance the utility of KLF2 antibodies in single-cell analysis of heterogeneous tissues?

Several emerging techniques promise to enhance KLF2 antibody applications in single-cell analysis:

• Mass cytometry (CyTOF): Converting FITC-conjugated KLF2 antibodies to metal-tagged versions would allow integration into high-dimensional CyTOF panels (30-40 parameters simultaneously), enabling comprehensive phenotyping of KLF2-expressing cells within heterogeneous populations. This would be particularly valuable for analyzing complex tissues like kidney glomeruli during hypertensive stress.

• Single-cell RNA-seq coupled with protein detection (CITE-seq):

  • Combining transcriptome analysis with antibody-based protein detection

  • Would allow correlation of KLF2 protein levels with global gene expression patterns

  • Could help identify novel KLF2-regulated pathways in specific cell subsets

• Spatial transcriptomics with protein co-detection:

  • Technologies like 10x Visium with immunofluorescence overlay

  • Would preserve tissue architecture while providing transcriptome data

  • FITC-conjugated KLF2 antibodies could be used to correlate protein localization with spatially-resolved transcriptomes

• Proximity ligation assays (PLA):

  • Detect protein-protein interactions involving KLF2

  • Would help map KLF2 interactome in different cell types

  • Could reveal how interactions change under conditions like hypertensive stress

• Live-cell imaging with nanobody-based detection:

  • Development of anti-KLF2 nanobodies conjugated to FITC

  • Would enable real-time tracking of KLF2 dynamics in living cells

  • Could reveal temporal aspects of KLF2 regulation currently inaccessible with fixed-cell approaches

These advanced techniques would significantly enhance our understanding of how KLF2 expression heterogeneity within tissues contributes to pathophysiological processes in diseases like hypertensive nephropathy.

How might researchers effectively integrate KLF2 antibody-based detection with functional genomics approaches?

Integrating KLF2 antibody-based detection with functional genomics requires strategic methodological combinations:

• ChIP-seq followed by immunofluorescence validation:

  • Identify genome-wide KLF2 binding sites using chromatin immunoprecipitation sequencing

  • Validate cell type-specific binding with FITC-conjugated KLF2 antibodies

  • Correlate binding patterns with expression of target genes in the same cells

• CRISPR screening with antibody-based readouts:

  • Perform genome-wide CRISPR screens targeting regulators of KLF2

  • Use FITC-conjugated KLF2 antibodies in flow cytometry as a readout

  • Identify novel factors controlling KLF2 expression in endothelial cells or immune cells

• Single-cell multi-omics integration:

  • Combine single-cell ATAC-seq (for chromatin accessibility)

  • With index sorting using FITC-conjugated KLF2 antibodies

  • Link epigenetic states to KLF2 protein levels in individual cells

• Spatial multi-omics approaches:

  • Use FITC-conjugated KLF2 antibodies for spatial mapping

  • Integrate with spatial transcriptomics data

  • Map microenvironmental influences on KLF2 expression

• Perturbation biology with high-content imaging:

  • Systematic gene knockdown/overexpression screens

  • Quantitative imaging with FITC-conjugated KLF2 antibodies

  • Multiparametric analysis of KLF2 levels, localization, and associated phenotypes

This integration would be particularly valuable for understanding how KLF2 regulates endothelial responses to pressure, as demonstrated in hypertensive nephropathy models where KLF2 levels influence fibrosis markers like fibronectin and TGF-β .

What are the most promising therapeutic applications emerging from KLF2 research that could be monitored using antibody-based detection?

Emerging therapeutic applications from KLF2 research that could be monitored with antibody-based detection include:

• Statin-based therapies for endothelial protection:

  • Statins like simvastatin upregulate KLF2 expression

  • FITC-conjugated KLF2 antibodies could monitor treatment efficacy in preclinical models

  • Research has shown simvastatin (10 μM) reduces pressure-induced fibrosis in glomerular endothelial cells

  • Monitoring parameters would include:

    • KLF2 expression levels in target tissues

    • Reduction in fibrotic markers (fibronectin, TGF-β)

    • Changes in inflammatory signaling (IL-18 levels)

• Cell-specific KLF2 modulation in immune disorders:

  • KLF2 restrains CD4+ T follicular helper cell differentiation

  • Deletion increases antibody production and B cell responses

  • Therapeutic targeting could modulate antibody responses in autoimmunity

  • FITC-conjugated KLF2 antibodies would help monitor:

    • T cell subset-specific KLF2 expression

    • Effects on germinal center formation

    • Changes in pathogenic antibody production

• Angiotensin pathway modulation in hypertensive nephropathy:

  • KLF2's renoprotective effects are linked to regulation of angiotensin II and AT1R

  • AT1R blockers could be combined with KLF2-enhancing therapies

  • Flow cytometry with FITC-conjugated KLF2 antibodies could track:

    • Restoration of endothelial KLF2 expression

    • Reduction in endothelial apoptosis

    • Changes in AT1R expression levels

• Novel pressure-sensing pathway interventions:

  • Targeting molecular components linking mechanical stress to KLF2 downregulation

  • Could benefit patients with hypertensive organ damage

  • Antibody-based assays would monitor:

    • Pathway component modifications

    • Resulting changes in KLF2 levels

    • Functional outcomes in pressure-response models

These emerging therapeutic applications highlight the importance of KLF2 as both a biomarker and therapeutic target across cardiovascular, renal, and immune disorders, with antibody-based detection methods critical for translational research progress.