S100A7 Antibody, FITC conjugated

What is S100A7 protein and why is it a significant research target?

S100A7 (also known as Psoriasin) is a small calcium-binding protein of approximately 11.5 kDa that belongs to the S100 protein family. It has significant research importance because:

• It is highly expressed in squamous cell carcinomas (SCC) and is related to the terminal differentiation of keratinocytes

• It displays heterogeneous and inducible characteristics in various SCC types, including lung, esophagus, oral cavity, skin, cervix, and bladder

• It functions as a dual regulator in cancer, promoting proliferation while suppressing squamous differentiation

• It plays a crucial role in inflammatory responses and is upregulated in inflammatory skin conditions like psoriasis

• It interacts with the Receptor for Advanced Glycation End Products (RAGE) in a zinc-dependent manner to promote inflammation and cell migration

What are the optimal applications for FITC-conjugated S100A7 antibodies?

FITC-conjugated S100A7 antibodies are particularly valuable for the following applications:

• Flow cytometry (intracellular) - Allows for quantitative analysis of S100A7 expression across cell populations

• Immunofluorescence microscopy - Enables visualization of S100A7 localization within cells and tissues

• Double immunofluorescence staining - Permits co-localization studies with other proteins such as keratin-13

• Live cell imaging - FITC's spectral properties make it suitable for real-time visualization in certain experimental setups

Note: The optimal dilution for each application should be experimentally determined based on the specific antibody concentration and experimental conditions .

How do I perform proper sample preparation for S100A7 detection using a FITC-conjugated antibody?

For optimal detection of S100A7 using FITC-conjugated antibodies:

For cell lines:

• Culture cells on cover-glass slides

• Wash cells with PBS to remove media components

• Fix with 4% buffered paraformaldehyde for 10-15 minutes at room temperature

• Permeabilize with 0.5% Triton X-100 for 10 minutes (for intracellular detection)

• Block with 3% BSA-PBS solution for 30-60 minutes

• Incubate with primary antibody at the optimized dilution (typically 1 μg/mL for 30 minutes at room temperature)

• Wash thoroughly to remove unbound antibody

• Counterstain nuclei with DAPI if needed

• Mount and analyze under a fluorescence microscope

For tissue sections:

• Process tissues following standard protocols for frozen or paraffin sections

• For paraffin sections, perform antigen retrieval (10 mM sodium citrate pH 6.0 or 1 mM EDTA pH 8.0 in a pressure cooker for 40 minutes)

• Follow the same blocking and staining steps as for cell lines

How can I design experiments to investigate the relationship between S100A7 expression and cancer progression using FITC-conjugated antibodies?

To investigate the relationship between S100A7 expression and cancer progression:

Experimental Design Framework:

• Multi-parameter flow cytometry analysis:

  • Use FITC-conjugated S100A7 antibody alongside other markers for cell cycle (PI), stemness (CD44), and differentiation (Keratins)

  • Analyze correlation between S100A7 levels and these parameters

  • Compare expression in primary tumors versus metastatic sites

• Tissue microarray (TMA) analysis:

  • Create TMAs containing samples from different stages of cancer progression

  • Perform immunofluorescence with FITC-conjugated S100A7 antibody

  • Quantify expression levels using image analysis software

  • Correlate with clinicopathological parameters

• In vitro modulation studies:

  • Establish cell lines with controlled S100A7 expression (overexpression/knockdown)

  • Monitor changes in proliferation, migration, and differentiation

  • Use FITC-conjugated antibody to confirm expression levels and localization

Validation Strategy:

• Confirm antibody specificity using Western blot and S100A7 knockout/knockdown controls

• Include multiple cancer types to establish pattern specificity

• Validate findings with patient-derived xenograft models

Research has shown that S100A7 expression is heterogeneous in SCC tissues and increases with disease progression, suggesting its potential role as a prognostic marker .

What methodological challenges might I encounter when using FITC-conjugated S100A7 antibodies for co-localization studies, and how can I overcome them?

Common Challenges and Solutions:

For co-localization studies of S100A7 with other proteins (like keratin-13), a validated protocol involves:

• Sequential staining with S100A7 antibody followed by TRITC-labeled secondary

• Blocking again with BSA

• Staining with second primary antibody

• Using a differently labeled secondary antibody (e.g., FITC-labeled anti-mouse IgG)

• Counterstaining nuclei with DAPI

How can I optimize flow cytometry protocols for intracellular detection of S100A7 using FITC-conjugated antibodies?

Optimized Flow Cytometry Protocol:

• Cell Preparation:

  • Harvest cells (1-5 × 10^6 cells per sample)

  • Wash twice in PBS

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

• Permeabilization Options:

  • For standard permeabilization: 0.5% Triton X-100 for 10 minutes

  • For gentle permeabilization: 0.1% saponin in PBS with 0.09% sodium azide

• Blocking and Staining:

  • Block with 3% BSA for 30 minutes

  • Incubate with FITC-conjugated S100A7 antibody at optimized concentration

  • Based on literature, start with 1 μg/mL and titrate as needed

  • Incubate for 30 minutes at room temperature in the dark

• Controls (Critical for Method Validation):

  • Include isotype control (FITC-conjugated non-immune IgG)

  • Use positive control (cell line with known S100A7 expression, e.g., HCC94 cells)

  • Use negative control (cell line with minimal S100A7 expression, e.g., A-431 cells under standard culture conditions)

• Analysis Parameters:

  • Gate on intact cells based on FSC/SSC

  • Exclude doublets using FSC-H vs FSC-A

  • Set PMT voltage based on negative control

  • Analyze minimum 10,000 events per sample

Troubleshooting Guidance:

• If signal is weak: increase antibody concentration, extend incubation time, optimize permeabilization

• If background is high: use more stringent washing, reduce antibody concentration, optimize blocking

• If cell clumping occurs: filter samples through 40 μm cell strainer before analysis

What are the current controversies or contradictions in S100A7 research, and how can FITC-conjugated antibodies help resolve them?

Several controversies exist in S100A7 research that FITC-conjugated antibodies could help address:

Controversy 1: Dual role in cancer progression

• Some studies report S100A7 promotes cancer cell proliferation

• Other studies suggest it suppresses differentiation and invasiveness

• Resolution approach: Use FITC-conjugated S100A7 antibodies for live-cell imaging to track S100A7 expression during cell cycle progression and cellular differentiation in real-time

Controversy 2: Intracellular localization and function

• Studies report variable localization (cytoplasmic, nuclear, membrane-associated)

• Function may differ based on subcellular localization

• Resolution approach: Combine FITC-conjugated S100A7 antibodies with organelle-specific markers for high-resolution confocal microscopy to definitively map localization patterns across different cell types and conditions

Controversy 3: S100A7 vs. S100A15 distinction

• S100A7 and S100A15 are highly homologous proteins with potentially distinct functions

• Many studies fail to distinguish between them

• Resolution approach: Develop highly specific FITC-conjugated antibodies with validated specificity for S100A7 vs. S100A15; use in parallel experiments to characterize differential expression and function

Controversy 4: RAGE-dependent vs. RAGE-independent signaling

• Some studies show S100A7 signals through RAGE

• Others suggest alternative receptors

• Resolution approach: Use FITC-conjugated S100A7 antibodies in receptor blocking experiments with simultaneous visualization of downstream signaling events

What are the critical factors affecting sensitivity and specificity when using FITC-conjugated S100A7 antibodies?

Several factors can significantly impact the performance of FITC-conjugated S100A7 antibodies:

Antibody Source and Validation:

• Clone selection is critical - different clones may recognize different epitopes

• Validated clones for S100A7 detection include 47C1068 and 128

• Recombinant monoclonal antibodies generally provide higher consistency than polyclonal antibodies

Technical Factors Affecting Performance:

Recommendations for Maximizing Signal-to-Noise Ratio:

• Store FITC-conjugated antibodies in the dark at 4°C to prevent photobleaching

• Include proper negative controls (isotype and unstained)

• Titrate antibody concentration for each application

• Use freshly prepared samples when possible

• Consider multi-parameter analysis to correlate S100A7 expression with other markers

How can I quantitatively analyze S100A7 expression patterns in heterogeneous tissue samples?

S100A7 shows significant heterogeneity in expression patterns, particularly in squamous cell carcinomas . To quantitatively analyze this heterogeneity:

Image Acquisition and Analysis Pipeline:

• Standardized Image Acquisition:

  • Use consistent exposure settings across all samples

  • Capture multiple fields per sample (minimum 5-10 random fields)

  • Include calibration standards for fluorescence intensity normalization

• Multi-level Scoring System:

  • Implement scoring system similar to established protocols:

    • 0: No positive cells

    • 1: <10% cancer cells positive

    • 2: >10% and <50% cancer cells positive

    • 3: >50% and <75% cancer cells positive

    • 4: >75% cancer cells positive

• Subcellular Localization Analysis:

  • Score separately for cytoplasmic, nuclear, and membrane localization

  • Quantify co-localization with differentiation markers like keratin-13

• Digital Image Analysis:

  • Use software (ImageJ, CellProfiler, QuPath) for unbiased quantification

  • Apply threshold-based segmentation to identify positive cells

  • Extract parameters including:

    • Mean fluorescence intensity

    • Percentage of positive cells

    • Subcellular distribution patterns

    • Correlation with differentiation or tumor grade

• Statistical Analysis:

  • Apply appropriate statistical tests for heterogeneous data

  • Consider hierarchical clustering to identify expression patterns

  • Correlate with clinicopathological data when available

This quantitative approach has been validated in research showing that S100A7 expression patterns correlate with the degree of differentiation in multiple SCC types .

How do I design experiments to investigate S100A7-RAGE interaction using FITC-conjugated antibodies?

The interaction between S100A7 and RAGE (Receptor for Advanced Glycation End products) is a critical mechanism mediating S100A7's pro-inflammatory and pro-tumorigenic effects . To study this interaction:

Experimental Design Framework:

• Co-localization Studies:

  • Use FITC-conjugated S100A7 antibody together with a differently labeled RAGE antibody

  • Analyze co-localization in:

    • Cancer cell lines with varying RAGE expression

    • Tissue sections from inflammatory conditions

    • Zinc-supplemented vs. zinc-depleted conditions (as S100A7-RAGE binding is zinc-dependent)

• Functional Analysis of S100A7-RAGE Interaction:

  • Treat cells with recombinant S100A7 protein

  • Block RAGE using FPS-ZM1 (RAGE inhibitor)

  • Monitor outcomes using:

    • Calcium flux assays

    • ERK/AKT/STAT3 phosphorylation

    • Cell migration assays

    • Angiogenesis assays

• Flow Cytometry-Based Binding Assays:

  • Use FITC-conjugated S100A7 to detect binding to RAGE-expressing cells

  • Compete with unlabeled S100A7 or RAGE antibodies

  • Test binding in presence/absence of zinc

  • Analyze by flow cytometry for quantitative binding assessment

• Proximity Ligation Assay:

  • Combine FITC-conjugated S100A7 antibody with RAGE antibody

  • Use proximity ligation to generate fluorescent signal when proteins are in close proximity

  • Quantify interaction under various conditions

Key Controls and Validations:

• Use cells with RAGE knockdown/knockout

• Include competitive binding with unlabeled proteins

• Test specificity with other S100 family members

• Verify with alternative techniques (co-immunoprecipitation, FRET)

What are common troubleshooting issues with FITC-conjugated S100A7 antibodies and their solutions?

Below is a comprehensive troubleshooting guide for issues commonly encountered when working with FITC-conjugated S100A7 antibodies:

Special Considerations for S100A7:

• Inducible expression: S100A7 expression can be induced in certain conditions; negative results in standard culture may not reflect potential expression

• Subcellular localization variability: S100A7 can be found in cytoplasm, nucleus, or membrane; ensure your protocol can detect all relevant localizations

• Differentiation-dependent expression: Expression often correlates with differentiation state; consider analyzing cells at different densities/differentiation stages

How can I optimize double immunofluorescence protocols to study S100A7 co-localization with differentiation markers?

Optimizing double immunofluorescence staining for S100A7 and differentiation markers (like keratin-13) requires careful consideration of antibody compatibility and staining sequence:

Optimized Protocol for Double Immunofluorescence:

• Sample Preparation:

  • Fix cells/tissues with 4% paraformaldehyde for 15 minutes

  • Permeabilize with 0.5% Triton X-100 for 10 minutes

  • Block with 3% BSA-PBS solution for 1 hour at room temperature

• Sequential Staining Approach (Recommended):

  • First Primary: Apply unconjugated S100A7 antibody (1:100-1:500 dilution)

  • Incubate for 1 hour at 37°C

  • Wash thoroughly with PBS (3 × 5 minutes)

  • First Secondary: Apply TRITC-labeled secondary antibody (1:200-1:500)

  • Incubate for 1 hour at 37°C

  • Wash thoroughly with PBS (3 × 5 minutes)

  • Re-block with 3% BSA-PBS

  • Second Primary: Apply differentiation marker antibody (e.g., keratin-13)

  • Incubate for 1 hour at 37°C

  • Wash thoroughly with PBS (3 × 5 minutes)

  • Second Secondary: Apply FITC-labeled secondary antibody (1:200-1:500)

  • Incubate for 1 hour at 37°C

  • Wash thoroughly with PBS (3 × 5 minutes)

  • Counterstain nuclei with DAPI

  • Mount with antifade medium

• Alternative Approach with Directly Conjugated Antibodies:

  • Apply FITC-conjugated S100A7 antibody

  • Wash thoroughly

  • Apply differently conjugated differentiation marker antibody (e.g., PE-conjugated)

  • Complete washing and mounting as above

Critical Optimization Steps:

• Determine optimal antibody dilutions for each primary antibody

• Test different fixation methods if epitope masking is suspected

• Validate staining specificity with appropriate controls

• Optimize microscope settings for minimal crosstalk between channels

Research has shown that S100A7 and keratin-13 show similar staining patterns in HCC94 cells, suggesting co-regulation during differentiation . This protocol allows for detailed analysis of this relationship.

What considerations should be taken when designing experiments to study S100A7 induction in different experimental conditions?

S100A7 shows significant inducibility in various experimental conditions . When designing experiments to study this induction:

Experimental Design Framework:

• Baseline Characterization:

  • Screen cell lines for basal S100A7 expression

  • Categorize as high expressors (e.g., HCC94) or low expressors (e.g., FaDu, A-431)

  • Document heterogeneity within populations using FITC-conjugated S100A7 antibody and flow cytometry

• Induction Conditions to Test:

  Condition Type	Specific Stimuli	Monitoring Timeline	Detection Method
  Growth conditions	Suspension culture, Cell density variation	24-72 hours	Flow cytometry with FITC-conjugated S100A7 antibody
  Inflammatory mediators	IL-6, IL-17, TNF-α	6-48 hours	Flow cytometry and Western blot
  Growth factors	IGF-1 , EGF	12-72 hours	Flow cytometry and Western blot
  Differentiation agents	Calcium, Serum	3-7 days	Immunofluorescence
  Stress conditions	Hypoxia, Oxidative stress	6-24 hours	Flow cytometry

• Analysis of Signaling Pathways:

  • Monitor activation of known regulatory pathways:

    • STAT3 pathway (shown to regulate S100A7 expression)

    • MAPK pathway (ERK, JNK, p38)

    • AKT pathway

  • Use pathway inhibitors to confirm mechanistic relationships

• Parallel Analysis of Differentiation Markers:

  • Monitor keratin-4, keratin-13, TG-1, and involucrin alongside S100A7

  • Correlate S100A7 induction with differentiation status

• In Vivo Validation:

  • Establish xenograft models with inducible S100A7 expression

  • Use FITC-conjugated antibodies for tissue analysis

Critical Considerations:

• Include time-course analysis to capture dynamics of induction

• Use multiple detection methods (flow cytometry, immunofluorescence, Western blot)

• Consider heterogeneity - analyze both population averages and single-cell distributions

• Include relevant controls (positive, negative, vehicle)

This approach has been validated in studies showing that S100A7-positive cells can be induced in HCC94, FaDu, and A-431 cells both in vitro and in vivo .

How can FITC-conjugated S100A7 antibodies be used in multiplexed imaging systems to better understand tumor microenvironment?

Multiplexed imaging with FITC-conjugated S100A7 antibodies offers powerful insights into the tumor microenvironment:

Advanced Multiplexing Approaches:

• Conventional Multiplexed Immunofluorescence:

  • Combine FITC-conjugated S100A7 antibody with antibodies against:

    • Immune cell markers (CD4, CD8, CD68)

    • Cancer stem cell markers (CD44, ALDH)

    • Differentiation markers (Keratins)

    • Proliferation markers (Ki67)

  • Use spectrally distinct fluorophores (TRITC, Cy5, Cy7)

  • Analyze with multispectral imaging systems

• Cyclic Immunofluorescence (CycIF):

  • Perform iterative rounds of staining, imaging, and antibody stripping

  • Include FITC-conjugated S100A7 antibody in appropriate round

  • Build multidimensional dataset with 10-40 markers on the same section

  • Computationally reconstruct to analyze spatial relationships

• Mass Cytometry Imaging (IMC):

  • Label S100A7 antibody with rare earth metals instead of FITC

  • Combine with 30+ additional markers

  • Analyze spatial distribution at subcellular resolution

• Spatial Transcriptomics Integration:

  • Combine FITC-conjugated S100A7 antibody imaging with spatial transcriptomics

  • Correlate protein expression with transcriptional programs

  • Identify microenvironmental factors driving S100A7 expression

Research Applications:

• Map S100A7 expression relative to immune infiltrates (S100A7 interacts with RAGE to promote immune cell migration)

• Analyze relationship between S100A7 and angiogenesis markers (S100A7 promotes angiogenesis)

• Investigate correlation between S100A7 expression and cancer stem cell populations

• Study spatial heterogeneity of S100A7 expression within tumors and its relationship to microenvironmental factors

This approach leverages the observation that S100A7 expression shows significant heterogeneity in tumors and may interact with various components of the tumor microenvironment .

What are emerging applications of S100A7 antibodies in personalized medicine research?

Emerging applications of S100A7 antibodies in personalized medicine include:

Diagnostic and Prognostic Applications:

• Patient Stratification: S100A7 expression patterns may identify patient subgroups with different prognosis or treatment responses

• Liquid Biopsy Development: Detection of circulating S100A7 protein as a potential biomarker

• Predictive Biomarker Research: Studies suggest S100A7 overexpression correlates with poor prognosis in several cancer types

Therapeutic Target Identification:

• Anti-RAGE Therapy Response Prediction: S100A7-RAGE interaction is a potential therapeutic target; antibody-based detection could identify patients likely to respond

• Differentiation Therapy Monitoring: Since S100A7 is linked to differentiation status, monitoring its expression could help assess response to differentiation-inducing therapies

• Combination Therapy Design: S100A7 inhibits squamous differentiation while promoting proliferation; targeting this dual function might enhance existing therapies

Methodological Approaches:

• Develop tissue microarray (TMA) screening protocols using FITC-conjugated S100A7 antibodies

• Establish standardized reporting systems for S100A7 expression patterns

• Create multiplexed panels including S100A7 and other prognostic markers

• Develop companion diagnostic assays for therapies targeting S100A7-mediated pathways

Validation Requirements:

• Large-scale, multi-center studies correlating S100A7 expression with clinical outcomes

• Standardized detection protocols to ensure reproducibility

• Integration with other molecular and clinical parameters

The dual regulatory role of S100A7 in promoting proliferation while suppressing differentiation makes it a particularly interesting target for personalized medicine approaches in cancer treatment.

What novel research directions are emerging in the study of S100A7 that could benefit from FITC-conjugated antibodies?

Several cutting-edge research directions are emerging in S100A7 biology that could be advanced using FITC-conjugated antibodies:

1. S100A7 in Tumor Immune Microenvironment:

• Investigate how S100A7 shapes immune cell recruitment and activation through RAGE interaction

• Map spatial relationships between S100A7-expressing tumor cells and immune infiltrates

• Study how S100A7 expression correlates with immunotherapy response

• Methodological approach: Use FITC-conjugated S100A7 antibodies in multiplexed immunofluorescence panels with immune cell markers

2. S100A7 in Cancer Stem Cell Biology:

• Examine relationship between S100A7 expression and cancer stem cell markers

• Investigate whether S100A7-positive cells have enhanced tumorigenic potential

• Study how S100A7 expression changes during epithelial-to-mesenchymal transition

• Methodological approach: Combine FITC-conjugated S100A7 antibodies with flow cytometry sorting and functional assays

3. Extracellular Functions of S100A7:

• Characterize secreted S100A7 in the tumor microenvironment

• Study how secreted S100A7 affects neighboring cells

• Develop strategies to detect and quantify extracellular S100A7

• Methodological approach: Use FITC-conjugated antibodies for tracking secreted protein

4. S100A7 in Therapeutic Resistance:

• Investigate how S100A7 expression changes in response to therapies

• Determine if S100A7-positive cells show differential sensitivity to treatments

• Study whether S100A7 inhibition can overcome resistance

• Methodological approach: Use FITC-conjugated antibodies to monitor expression changes during treatment

5. S100A7 and S100A15 Distinction:

• Develop methods to clearly distinguish between these highly homologous proteins

• Characterize their potentially distinct functions

• Map their differential expression in tissues

• Methodological approach: Develop highly specific FITC-conjugated antibodies that can distinguish between these closely related proteins

These emerging directions highlight the continuing importance of S100A7 as a research target and the value of well-validated FITC-conjugated antibodies in advancing our understanding of its complex biology.