ARSD Antibody

What is ARSD and why would researchers develop antibodies against it?

ARSD (Arylsulfatase D) is a protein belonging to the sulfatase family, situated on the X chromosome alongside other aromatic sulfate enzymes with similar characteristics . It displays remarkably context-dependent functions in cancer biology, making it a valuable target for research antibodies.

Conversely, in glioma, ARSD promotes cancer development by regulating the immune microenvironment and JAK2/STAT3 signaling pathway, with high expression associated with shorter survival times . This dual role makes ARSD antibodies essential for studying:

• Tissue-specific expression patterns

• Subcellular localization

• Protein-protein interactions

• Prognostic significance

• Signaling pathway involvement

What cellular localization patterns should researchers consider when selecting an ARSD antibody?

Based on research findings, ARSD exhibits distinct localization patterns that researchers must consider when selecting antibodies:

In MCF-7 breast cancer cells, robust ARSD immunoreactivity is observed uniformly in the cytoplasm . This cytoplasmic distribution appears consistent in luminal subtype breast cancer tissues, while triple-negative breast cancer tissues show minimal expression .

When selecting an ARSD antibody, researchers should consider:

• Primary cytoplasmic detection capabilities

• Potential cross-reactivity with other sulfatase family members

• Application versatility (IHC, ICC, WB) for correlating localization with expression

• Validation in specific experimental models, as ARSD may localize differently in various cancer types

• Sensitivity for detecting the range of expression levels observed across different tissues

How do ARSD expression patterns differ across cancer types, and what implications does this have for antibody selection?

Research demonstrates significant variation in ARSD expression across cancer types:

qRT-PCR and Western blotting across breast cancer cell lines confirm that highly invasive cell lines (MDA-MB-231, BT-549) show lower ARSD expression, while ER-positive lines (MCF-7, T47D) demonstrate higher expression .

For antibody selection, these expression patterns necessitate:

• Appropriate sensitivity calibration based on expected expression levels

• Validation using positive controls from tissues known to express ARSD

• Standardized protocols for comparative studies across cancer types

• Concentration optimization to ensure detection across the spectrum of expression levels

What validation methods should be implemented for ARSD antibodies in Western blotting applications?

For rigorous validation of ARSD antibodies in Western blotting applications, researchers should implement the following methods:

• Positive and negative control cell lines:

  • Use MCF-7 and T47D (high ARSD expression) as positive controls

  • Use MDA-MB-231 and BT-549 (low ARSD expression) as negative or low-expression controls

  • Include HEK293T cells as a reference cell line as used in published studies

• Genetic validation:

  • Test the antibody in cells with ARSD knockdown (using siRNA or CRISPR)

  • Verify in cells with ARSD overexpression (as constructed in referenced studies)

  • Confirm expected band intensity changes correlate with genetic manipulation

• Molecular weight verification:

  • Verify expected molecular weight based on amino acid sequence

  • Check for potential post-translational modifications affecting migration

  • Use molecular weight markers appropriate for the expected size range

• Sample preparation optimization:

  • Test multiple lysis buffers (RIPA, NP-40, Triton X-100)

  • Evaluate the impact of protease and phosphatase inhibitors

  • Determine optimal protein loading amounts (typically 20-50μg)

Research shows that ARSD protein detection through Western blotting correlates with functional effects, as ARSD overexpression markedly inhibits migration, colony formation, and invasion of TNBC cells .

How can immunohistochemical protocols be optimized for detecting ARSD in different breast cancer subtypes?

Optimizing immunohistochemical protocols for ARSD detection requires tailored approaches for different breast cancer subtypes:

• Antigen retrieval optimization:

  • For ER+ tissues: Heat-induced epitope retrieval using citrate buffer (pH 6.0), 20 minutes

  • For TNBC tissues: More aggressive retrieval using Tris-EDTA buffer (pH 9.0), 25-30 minutes

  • Consider dual retrieval approaches for difficult samples

• Antibody concentration and incubation parameters:

  • For high-expressing luminal subtypes: 1:200-1:500 dilution, 1-hour incubation

  • For low-expressing TNBC: 1:50-1:100 dilution, overnight incubation at 4°C

  • Test multiple antibody clones if initial results are suboptimal

• Signal detection systems:

  • For ER+ tissues: Standard HRP-polymer detection systems provide sufficient sensitivity

  • For TNBC: Implement tyramide signal amplification for enhanced detection

  • Consider chromogenic multiplex IHC to co-detect ARSD with ER/PR/HER2

The importance of protocol optimization is highlighted by research showing "strong positive expression of ARSD observed in luminal subtype BC tissues, while it was hard to find positive signals in TNBC tissues" . This differential expression makes standardized protocols essential for accurate subtype comparisons.

What approaches should researchers use to investigate ARSD's role in the Hippo/YAP pathway in breast cancer?

To investigate ARSD's role in Hippo/YAP pathway activation, researchers should implement a comprehensive antibody-based strategy:

• Multiplex co-detection approach:

  • Use ARSD antibodies compatible with multiplex immunofluorescence

  • Co-stain for YAP and phospho-YAP to determine activation status

  • Implement nuclear/cytoplasmic fractionation to track YAP localization

  • Correlate ARSD expression with YAP nuclear exclusion

• Functional validation using genetic manipulation:

  • In ARSD-overexpression models, assess:

    • YAP phosphorylation status

    • YAP subcellular localization

    • Target gene expression (CTGF, CYR61)

  • In ARSD-knockdown models, evaluate:

    • Effects on YAP-dependent transcription

    • Cellular phenotypes (proliferation, migration)

• Protein interaction analysis:

  • Perform co-immunoprecipitation with anti-ARSD antibodies

  • Identify Hippo pathway components in the precipitated complex

  • Confirm interactions with reverse IP experiments

  • Validate with proximity ligation assays to visualize interactions in situ

Research demonstrates that ARSD overexpression significantly suppresses the proliferation of MDA-MB-231 and BT-549 cells compared to control cells, potentially through Hippo/YAP pathway modulation .

How should researchers investigate the contradictory roles of ARSD in breast cancer versus glioma?

The contradictory roles of ARSD in breast cancer (tumor suppressive) versus glioma (tumor promoting) necessitate carefully designed comparative investigations:

• Parallel expression analysis:

  • Use identical ARSD antibody clones and protocols across cancer types

  • Implement tissue microarrays containing both cancer types

  • Quantify expression using standardized digital pathology algorithms

  • Correlate with patient outcomes in both cancer types

• Pathway-specific mechanistic investigation:

  • In breast cancer models:

    • Focus on ARSD's relationship with ERα, FOXA1, and GATA3

    • Examine Hippo/YAP pathway activation

  • In glioma models:

    • Investigate JAK2/STAT3 pathway effects

    • Assess immune cell infiltration patterns, particularly M2 macrophages

• Context-dependent interactome mapping:

  • Perform comparative immunoprecipitation with anti-ARSD antibodies in both cancer types

  • Identify differential binding partners through mass spectrometry

  • Validate cancer-specific interactions through targeted co-IP experiments

Research shows that ARSD expression is positively correlated with immunosuppressive cells (M2 macrophages, neutrophils, and Th2 cells) in glioma , while in breast cancer, it correlates with favorable prognosis and inhibits proliferation and migration .

What strategies should be employed for studying ARSD's interaction with transcription factors ERα, FOXA1, and GATA3?

To investigate ARSD's interaction with transcription factors ERα, FOXA1, and GATA3, researchers should implement these comprehensive strategies:

• Chromatin immunoprecipitation approach:

  • Use ChIP-validated antibodies for all four proteins

  • Implement sequential ChIP (ChIP-reChIP) to confirm co-occupancy

  • Focus on the ARSD promoter region from -2064 to +64 bp containing dense clusters of binding sites

  • Design primers targeting specific binding regions identified in published research

• Transcriptional regulation analysis:

  • Employ luciferase reporter assays to quantify transcriptional activation

  • Mutate predicted binding sites to confirm functional significance

  • Correlate binding with activation using RT-qPCR for ARSD expression

  • Implement CRISPR-based editing of identified binding sites

• Protein complex analysis:

  • Perform co-immunoprecipitation with antibodies against each factor

  • Detect ARSD using Western blotting in immunoprecipitated complexes

  • Visualize interactions using proximity ligation assays in intact cells

  • Map interaction domains through deletion mutant analysis

Research confirms these interactions, as "chromatin immunoprecipitation (ChIP) assay confirmed these bindings along with ERα, FOXA1, and GATA3 antibodies pull-down" .

How can phospho-specific ARSD antibodies be developed to study its role in the JAK2/STAT3 pathway?

Developing phospho-specific antibodies for ARSD in the JAK2/STAT3 pathway requires a systematic approach:

• Phosphorylation site identification:

  • Perform in silico analysis to identify JAK2 consensus sequences in ARSD

  • Conduct phosphoproteomic analysis of ARSD in glioma cells with active JAK2/STAT3 signaling

  • Prioritize sites that show dynamic phosphorylation upon pathway modulation

• Immunogen design and antibody production:

  • Synthesize phosphopeptides corresponding to identified sites

  • Generate both phosphorylated and non-phosphorylated peptide versions

  • Immunize rabbits using extended protocols for phospho-specific antibodies

  • Screen antisera for phospho-specificity using ELISA and Western blotting

• Validation in disease models:

  • Test antibodies in glioma cells with modulated JAK2/STAT3 activity:

    • JAK2 inhibitor treatment (should reduce ARSD phosphorylation)

    • IL-6 stimulation (should increase ARSD phosphorylation)

  • Perform phosphatase treatments on lysates as specificity controls

  • Validate in ARSD-knockout models as negative controls

• Application to clinical samples:

  • Assess phospho-ARSD levels in glioma patient samples

  • Correlate with STAT3 activation markers

  • Evaluate relationship with immune cell infiltration, particularly M2 macrophages

  • Analyze association with patient outcomes

This approach would enable detailed investigation of ARSD's role in the JAK2/STAT3 pathway in glioma, where research has shown it "can promote glioma development by regulating immune microenvironment and JAK2/STAT3 signaling pathway" .

What fixation and antigen retrieval methods should be optimized for ARSD detection in tissue samples?

For optimal ARSD detection in tissue samples, researchers should implement these evidence-based protocols:

• Fixation parameters:

  • Optimal fixation: 10% neutral-buffered formalin for 24-48 hours

  • For fresh-frozen sections: Brief post-fixation (10 minutes) in 4% paraformaldehyde

  • Avoid extended fixation periods that may mask ARSD epitopes

  • Process tissues using standard dehydration and paraffin embedding protocols

• Antigen retrieval optimization:

  • Primary recommendation: Heat-induced epitope retrieval (HIER)

  • Buffer options:

    • First choice: 10mM sodium citrate buffer (pH 6.0)

    • Alternative: Tris-EDTA buffer (pH 9.0) for difficult samples

  • Heating parameters:

    • Pressure cooker: 125°C, 3 minutes

    • Microwave: 95-98°C, 20 minutes

    • Water bath: 95°C, 30 minutes

• Section handling considerations:

  • Optimal section thickness: 4-5μm

  • Mount on positively charged slides

  • Include drying step (37°C overnight) to prevent tissue loss

  • Perform antigen retrieval within 24-48 hours of sectioning

These protocols align with successful ARSD detection in published studies, where immunohistochemical staining clearly differentiated between luminal breast cancer tissues (strong positive expression) and TNBC tissues (minimal signal) .

How should inconsistent ARSD antibody staining between tissue preparation methods be addressed?

Addressing inconsistent ARSD staining between fresh-frozen and FFPE tissues requires systematic troubleshooting:

• Epitope sensitivity assessment:

  • Test multiple antibody clones targeting different ARSD regions

  • Create comparative data for each preparation method:

• Protocol adaptation strategies:

  • For FFPE tissues with weak signals:

    • Extend antigen retrieval time (20-30 minutes)

    • Implement higher antibody concentrations (2-3× that used for frozen)

    • Utilize signal amplification systems (tyramide-based)

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

  • For fresh-frozen tissues with high background:

    • Add brief post-sectioning fixation step

    • Increase blocking stringency (5% serum + 0.3% Triton X-100)

    • Reduce antibody concentration by 50%

    • Include additional washing steps with 0.1% Tween-20

• Validation approach:

  • Process adjacent sections using both methods

  • Include appropriate positive controls (ER+ breast cancer for ARSD)

  • Implement orthogonal detection methods (RNA-ISH for ARSD mRNA)

This systematic approach ensures reliable ARSD detection across preparation methods, critical for comparative studies between different cancer types.

What controls are essential for ARSD antibody-based prognostic studies?

For rigorous prognostic studies using ARSD antibodies, these essential controls must be implemented:

• Antibody validation controls:

  • Positive tissue controls: ER-positive breast cancer tissue (MCF-7 xenografts or validated clinical samples)

  • Negative tissue controls: Triple-negative breast cancer tissue or validated negative samples

  • Technical controls: Primary antibody omission, isotype-matched IgG controls

  • Specificity controls: Pre-incubation with immunizing peptide to block specific binding

• Reference standards:

  • Expression gradient controls: Include samples with known high (MCF-7), medium (SKBR3), and low (MDA-MB-231) ARSD expression

  • Internal tissue controls: Normal adjacent tissue when available

  • Cell line standard curves: Embedded cell lines with defined ARSD expression levels

• Quantification quality controls:

  • Scoring system validation: Compare manual H-scores with automated quantification

  • Inter-observer controls: Multiple independent scorers evaluate subset of samples

  • Batch controls: Process reference sample with each batch of staining

  • Antibody lot testing: Validate each new lot against reference standards

• Clinical outcome controls:

  • Known prognostic marker controls: Process sections for established markers (e.g., ER, PR, HER2)

  • Treatment stratification: Control for treatment effects on ARSD expression

  • Survival reference points: Include patients with known outcomes at specific timepoints

What protocol should be used for ARSD detection in flow cytometry to study immune cell relationships?

For flow cytometric detection of ARSD in the context of immune cell studies, particularly in glioma models, implement this optimized protocol:

• Sample preparation:

  • Fresh tissue processing:

    • Mechanically dissociate tissue using a gentleMACS Dissociator

    • Enzymatically digest with collagenase D (1mg/ml) and DNase I (0.1mg/ml) for 30 minutes at 37°C

    • Filter through 70μm cell strainers

    • Perform RBC lysis if needed

• Surface marker staining:

  • Block Fc receptors with 5% normal serum (15 minutes, 4°C)

  • Stain with fluorochrome-conjugated antibodies for immune cell markers:

    • M2 macrophages: CD11b+/CD206+/CD163+

    • Neutrophils: CD11b+/Ly6G+

    • Th2 cells: CD4+/GATA3+

  • Wash twice with FACS buffer (PBS + 2% FBS + 1mM EDTA)

• ARSD intracellular staining:

  • Fix cells with 4% paraformaldehyde (10 minutes, room temperature)

  • Permeabilize with 0.1% saponin buffer

  • Block with 5% normal serum in permeabilization buffer

  • Stain with anti-ARSD antibody (optimized concentration)

  • Detect with fluorochrome-conjugated secondary antibody

  • Include phospho-STAT3 staining to assess pathway activation

• Multiparameter panel design:

• Essential controls:

  • Fluorescence Minus One (FMO) controls

  • Isotype controls matching primary antibody

  • ARSD-knockout or knockdown cells (negative control)

  • ARSD-overexpressing cells (positive control)

This protocol is designed based on research showing ARSD expression is "positively correlated with immunosuppressive cells, including M2 macrophages, neutrophils, and Th2 cells" in glioma .