HIST1H1C (Ab-167) Antibody

What is HIST1H1C and what cellular functions does it serve?

HIST1H1C, also known as Histone H1.2, Histone H1c, Histone H1d, or Histone H1s-1, is a linker histone that binds to DNA between nucleosomes, forming the macromolecular structure known as chromatin fiber. This protein is essential for the condensation of nucleosome chains into higher-order structured fibers. Beyond structural roles, HIST1H1C acts as a regulator of individual gene transcription through chromatin remodeling, nucleosome spacing, and DNA methylation . Research has demonstrated that HIST1H1C plays crucial roles in diverse cellular processes including epigenetic silencing, where it regulates both DNA methylation and histone H3 methylation at specific genetic loci such as H19 and Gtl2 in mouse embryonic stem cells .

What are the recommended applications for HIST1H1C (Ab-167) Antibody?

HIST1H1C (Ab-167) Antibody has been validated for multiple experimental applications with specific recommended dilutions:

The antibody has demonstrated reactivity against human, mouse, and rat HIST1H1C, making it suitable for comparative studies across these species . Researchers should optimize dilutions based on their specific experimental conditions and sample types.

What is the optimal sample preparation protocol for immunohistochemistry with HIST1H1C (Ab-167) Antibody?

For optimal immunohistochemistry results with HIST1H1C (Ab-167) Antibody, researchers should follow this validated protocol:

• Fix tissue sections appropriately (typically 4% paraformaldehyde)

• Perform antigen retrieval (heat-induced epitope retrieval in citrate buffer pH 6.0 is recommended)

• Block endogenous peroxidase activity with 3% H₂O₂

• Apply protein block (5% normal serum)

• Incubate with HIST1H1C (Ab-167) Antibody at 1:20-1:200 dilution overnight at 4°C (for mouse tissue) or room temperature (for human tissue arrays)

• Apply secondary antibody following manufacturer recommendations

• Visualize using 3,3'-diaminobenzidine substrate following ABC kit protocols

• Counterstain, dehydrate, and mount

For quantification, positively stained areas or cells can be analyzed using image analysis software such as ImagePro Plus based on four to six randomly selected fields per sample .

How can HIST1H1C (Ab-167) Antibody be used to study hepatocarcinogenesis mechanisms?

HIST1H1C has been implicated in hepatocarcinogenesis through regulation of signal transduction pathways. Researchers can use HIST1H1C (Ab-167) Antibody to investigate these mechanisms through multiple approaches:

• Tissue microarray analysis: The antibody has been successfully employed in human HCC tissue microarrays comprising tumor and paratumor tissues to assess HIST1H1C expression patterns in relation to other markers like phosphorylated STAT3 (p-STAT3 Y705) .

• Knockdown studies: Researchers can evaluate HIST1H1C function by comparing control cells with those transfected with shRNA targeting HIST1H1C, followed by antibody-based validation of knockdown efficiency through Western blot or immunostaining .

• Coimmunoprecipitation: HIST1H1C (Ab-167) Antibody can be used in co-IP experiments to investigate protein interactions, particularly with transcription factors like STAT3, which helps elucidate regulatory mechanisms in hepatocarcinogenesis .

• Chromatin immunoprecipitation (ChIP): The antibody enables investigation of HIST1H1C binding to specific gene promoters, illuminating its role in transcriptional regulation during liver cancer development .

When using HIST1H1C knockout mouse models in hepatocarcinogenesis studies, researchers should use this antibody to confirm the absence of the protein and correlate with phenotypic changes in tumor development and progression.

What methodological considerations are important when studying HIST1H1C's role in autophagy regulation?

HIST1H1C has been identified as a regulator of autophagy, particularly in the development of diabetic retinopathy. When investigating this relationship using HIST1H1C (Ab-167) Antibody, researchers should consider:

• Autophagy markers correlation: Combine HIST1H1C staining with autophagy markers such as ATG12-ATG5 complex, ATG7, ATG3, and LC3B-I to LC3B-II conversion. Evidence shows overexpression of HIST1H1C induces upregulation of these autophagy-related proteins .

• Flux analysis methodology: When studying autophagy flux, combine HIST1H1C (Ab-167) antibody detection with autophagy inhibitors (chloroquine, bafilomycin A1) treatment. Monitor SQSTM1/p62 levels alongside LC3B-I to LC3B-II conversion for accurate flux assessment .

• Cell viability correlation: Establish correlations between HIST1H1C expression, autophagy levels, and cell viability measures. Research indicates that HIST1H1C overexpression significantly reduces cell viability in retinal cells .

• Transcriptional analysis: Combine protein detection with transcriptional analysis of autophagy-related genes (Becn1, Atg12, Atg7, Atg5, Atg3, Map1lc3b), as HIST1H1C has been shown to regulate their expression .

• Stress response assessment: Evaluate how HIST1H1C levels affect cell responses to classic autophagy inducers (starvation, rapamycin) and disease-relevant stressors (high glucose), as HIST1H1C knockdown has been shown to inhibit stress-induced autophagy .

How can researchers validate HIST1H1C knockdown efficiency using Ab-167 antibody?

Validation of HIST1H1C knockdown is critical for functional studies. The following methodology using HIST1H1C (Ab-167) Antibody ensures comprehensive validation:

• Multi-level validation approach:

  • Transcriptional level: Perform qRT-PCR to quantify Hist1h1c mRNA

  • Protein level: Use Western blot with Ab-167 antibody (1:500-1:2000 dilution)

  • Cellular level: Perform immunofluorescence or immunohistochemistry (1:20-1:200 dilution)

• Western blot optimization: For optimal detection of knockdown efficiency:

  • Use nuclear fraction of cell lysates (as HIST1H1C is primarily nuclear)

  • Load 20-30 μg of protein per lane

  • Include appropriate loading controls (e.g., other nuclear proteins)

  • Quantify band intensity using densitometry and normalize to controls

• Time-course analysis: Validate knockdown at multiple time points post-transfection or treatment to ensure stable reduction throughout the experimental period.

Studies have shown significant decreases in both Hist1h1c mRNA and protein levels in shHist1h1c cells compared to control cells, with corresponding downregulation of Atg genes and proteins, demonstrating the effectiveness of this validation approach .

How can ChIP assays be optimized using HIST1H1C (Ab-167) Antibody?

Chromatin immunoprecipitation (ChIP) assays using HIST1H1C (Ab-167) Antibody require specific optimization strategies to study HIST1H1C binding to DNA regions:

• Cross-linking optimization: For linker histones like HIST1H1C, use 1% formaldehyde for 10 minutes at room temperature, as longer cross-linking may mask epitopes.

• Sonication parameters:

  • For HIST1H1C ChIP, aim for chromatin fragments of 200-500 bp

  • Use pulse sonication (30 seconds on/30 seconds off for 10-15 cycles)

  • Verify fragmentation via agarose gel electrophoresis before proceeding

• Antibody concentration: Use 3-5 μg of HIST1H1C (Ab-167) Antibody per ChIP reaction, which has been validated in previous studies examining HIST1H1C promoter binding .

• Region selection strategy: When designing primers for qPCR analysis of ChIP samples:

  • For human H1C promoter analysis, design primers covering three different regions ranging from -2000 bp to the transcription start site (TSS)

  • Include analysis of regions with predicted binding sites for interacting transcription factors like STAT3

• Controls and normalization:

  • Include IgG negative controls and positive controls (known HIST1H1C binding regions)

  • Normalize to input DNA (typically 1-10% of starting chromatin)

  • Express results as percent input or fold enrichment over IgG control

This optimized approach has been successfully employed to identify STAT3 binding sites within the promoters of human H1C and mouse H1c, shedding light on regulatory mechanisms .

What technical approaches are effective for studying HIST1H1C interactions with DNA methyltransferases?

Research has shown that some murine H1 subtypes, including HIST1H1C, interact with DNA methyltransferases DNMT1 and DNMT3B. To investigate these interactions using HIST1H1C (Ab-167) Antibody:

• Co-immunoprecipitation protocol optimization:

  • Use nuclear extracts (where both proteins primarily localize)

  • Pre-clear lysates with protein A/G beads to reduce background

  • Immunoprecipitate with 2-4 μg of HIST1H1C (Ab-167) Antibody

  • Perform reverse co-IP with DNMT1 and DNMT3B antibodies

  • Include appropriate controls (IgG, input, flow-through)

• Domain mapping experiments:

  • The C-terminal domain (CTD) of HIST1H1C is critical for interactions with DNMTs

  • Specifically, residues 167-221 are important for these interactions

  • Design experiments with chimeric constructs or truncated proteins to confirm binding regions

• Functional validation approaches:

  • Express HIST1H1C in cell lines and measure DNMT recruitment to specific loci

  • Assess DNA methylation changes at target regions (e.g., imprinting control regions)

  • Use bisulfite sequencing or methylation-specific PCR to quantify DNA methylation levels

• Competition assays:

  • Determine if HIST1H1C competes with other proteins (e.g., SET7/9 histone methyltransferase) for binding to specific genomic regions

  • Use ChIP-reChIP or sequential ChIP to assess co-occupancy or mutual exclusivity

These approaches have revealed that expression of HIST1H1C subtypes that interact with DNMT1 and DNMT3B leads to their recruitment and DNA methylation of the H19 and Gtl2 imprinting control regions, highlighting its role in epigenetic regulation .

How can researchers investigate HIST1H1C nuclear-cytoplasmic translocation in cellular stress conditions?

• Subcellular fractionation protocol:

  • Prepare nuclear and cytoplasmic fractions using established protocols

  • Validate fraction purity using markers (e.g., lamin for nucleus, GAPDH for cytoplasm)

  • Analyze HIST1H1C distribution by Western blot using Ab-167 antibody (1:500-1:2000)

  • Quantify relative amounts in each fraction across experimental conditions

• Immunofluorescence microscopy optimization:

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

  • Permeabilize with 0.1% Triton X-100 (5 minutes)

  • Block with 5% BSA or normal serum

  • Incubate with HIST1H1C (Ab-167) Antibody overnight at 4°C

  • Use high-resolution confocal microscopy to detect subtle changes in localization

  • Apply nuclear counterstaining (DAPI) and cytoplasmic/organelle markers

• Stimulus-dependent translocation analysis:

  • Compare normal conditions with stress conditions (high glucose, X-ray irradiation)

  • Include time-course analysis to capture dynamic translocation events

  • Quantify nuclear/cytoplasmic ratios using image analysis software

  • Present data as change in localization pattern over time

Research has shown that in retinal Müller cells (rMC-1) and 293T cells, HIST1H1C remains predominantly nuclear even under high glucose conditions, suggesting context-dependent translocation mechanisms .

What are common troubleshooting strategies for inconsistent HIST1H1C (Ab-167) Antibody staining in immunohistochemistry?

When encountering inconsistent staining results with HIST1H1C (Ab-167) Antibody in immunohistochemistry, researchers should systematically address:

• Fixation optimization:

  • Overfixation: Limit fixation time to 24 hours for optimal epitope preservation

  • Underfixation: Ensure complete tissue penetration by using appropriate fixative volume

  • Fixative type: 4% paraformaldehyde is recommended; formalin may cause excessive cross-linking

• Antigen retrieval modification:

  • Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

  • For heat-induced retrieval, compare citrate buffer (pH 6.0) vs. EDTA buffer (pH 9.0)

  • Optimize retrieval time (10-30 minutes) and temperature

• Antibody dilution titration:

  • Create a dilution series from 1:10 to 1:200 to determine optimal concentration

  • Include positive control tissue (such as lymphoid tissue) with known HIST1H1C expression

  • Develop standardized staining intensity scoring system

• Background reduction techniques:

  • Increase blocking time (1-2 hours with 5% normal serum)

  • Add 0.1-0.3% Triton X-100 to enhance antibody penetration

  • Include avidin/biotin blocking when using biotin-based detection systems

• Detection system optimization:

  • Compare ABC method vs. polymer-based detection systems

  • Adjust DAB development time (2-10 minutes) for optimal signal-to-noise ratio

  • Consider signal amplification for low-expressing samples

Studies using HIST1H1C (Ab-167) Antibody for human HCC tissue microarrays and mouse liver sections have demonstrated successful optimization using these approaches .

How can researchers address conflicting data between HIST1H1C protein levels and functional outcomes?

When experimental results show discrepancies between HIST1H1C protein levels and expected functional outcomes, consider these analytical approaches:

• Isoform-specific analysis:

  • HIST1H1C is one of several H1 variants (H1.1-H1.5)

  • Use primers and antibodies that discriminate between highly similar H1 subtypes

  • Perform comprehensive analysis of all H1 variants to identify potential compensatory mechanisms

• Post-translational modification assessment:

  • HIST1H1C function is regulated by phosphorylation, methylation, and other modifications

  • Use modification-specific antibodies alongside total HIST1H1C detection

  • Consider mass spectrometry to characterize modification patterns

• Functional domain isolation:

  • The C-terminal domain (CTD) of HIST1H1C (residues 167-221) is critical for many functions

  • Design experiments with chimeric constructs to isolate function-specific regions

  • Expression of H1c.d chimeric protein in which residues 167-221 are replaced can help localize functional differences

• Temporal analysis:

  • HIST1H1C effects may be delayed or require threshold accumulation

  • Design time-course experiments capturing protein levels and functional readouts

  • Consider pulsed expression or degradation systems to establish causality

• Context-dependent function assessment:

  • HIST1H1C functions differently across cell types and conditions

  • Compare results across multiple cell lines and primary cells

  • Test function under different stress conditions (starvation, rapamycin, high glucose)

This multifaceted approach has helped researchers resolve apparent contradictions, such as understanding why HIST1H1C remains nuclear in some cell types under conditions where it would translocate in others .

How can HIST1H1C (Ab-167) Antibody contribute to understanding epigenetic dysregulation in cancer?

HIST1H1C has emerging roles in cancer development through epigenetic mechanisms. Researchers can use HIST1H1C (Ab-167) Antibody to explore:

• Cancer-specific expression profiling:

  • The Cancer Genome Atlas data shows differential expression of H1.1-H1.5 in hepatocellular carcinoma

  • Use HIST1H1C (Ab-167) Antibody for tissue microarray analysis comparing multiple cancer types

  • Correlate HIST1H1C levels with clinical outcomes and molecular subtypes

• Epigenetic signature correlation:

  • Combine HIST1H1C immunostaining with DNA methylation analysis

  • Overlay HIST1H1C binding patterns with histone modification maps

  • Investigate relationships between HIST1H1C and cancer-specific epigenetic signatures

• Therapeutic response prediction:

  • Monitor HIST1H1C levels before and after epigenetic drug treatment

  • Determine if HIST1H1C patterns predict response to DNMT inhibitors or HDAC inhibitors

  • Develop HIST1H1C-based biomarkers for patient stratification

• Mechanistic pathway analysis:

  • Investigate HIST1H1C interaction with STAT3 signaling in cancer progression

  • Use promoter-reporter assays to study HIST1H1C regulation of oncogenes

  • Apply ChIP-seq with HIST1H1C (Ab-167) Antibody to map genome-wide binding patterns

This approach has revealed important connections between HIST1H1C and hepatocarcinogenesis, showing its potential as both a biomarker and therapeutic target in cancer research .

What methodological innovations can enhance the study of HIST1H1C in neurodegenerative conditions?

Recent research suggests HIST1H1C may play roles in neurodegenerative conditions through its effects on autophagy and inflammation. Researchers can advance this field using HIST1H1C (Ab-167) Antibody through:

• Brain region-specific expression mapping:

  • Optimize immunohistochemistry protocols for brain tissue

  • Map HIST1H1C expression across neuroanatomical regions in normal and diseased brains

  • Correlate with markers of neurodegeneration and neuroinflammation

• Cell type-specific function analysis:

  • Combine HIST1H1C staining with neuronal, astrocyte, and microglial markers

  • Use single-cell approaches to isolate cell type-specific effects

  • Apply conditional knockdown in specific neural cell populations

• Protein-protein interaction networks in neural context:

  • Identify neural-specific HIST1H1C interacting partners through co-IP with Ab-167

  • Map interaction changes during disease progression

  • Validate functional consequences through targeted disruption of interactions

• In vivo models bridging HIST1H1C and neurodegeneration:

  • Apply HIST1H1C (Ab-167) Antibody in established neurodegenerative disease models

  • Correlate HIST1H1C levels with autophagy markers and inflammatory cytokines

  • Test whether manipulating HIST1H1C levels affects disease progression

The foundation for this work comes from studies showing HIST1H1C regulates autophagy and inflammation in retinal cells, processes also critical in neurodegenerative conditions .