ch25hl1.1 Antibody

What is ch25hl1.1 and what is its function in zebrafish?

Ch25hl1.1 is a zebrafish (Danio rerio) gene that encodes a homolog of cholesterol 25-hydroxylase. This enzyme catalyzes the oxidation of cholesterol to 25-hydroxycholesterol (25HC) . In zebrafish, ch25hl1.1 is part of the antiviral response system. The gene is transiently upregulated during viral infections or following viral protein exposure, such as after SARS-CoV-2 spike protein injection . Ch25hl1.1 appears to be the main homolog responsible for antiviral 25HC production in zebrafish, as other ch25h homologs do not exhibit the same antiviral upregulation pattern .

Why are zebrafish models useful for studying ch25hl1.1 function?

Zebrafish provide an ideal vertebrate model for studying ch25hl1.1 function for several reasons:

• They combine favorable features of multiple model organisms: external embryo development allows easy visualization and manipulation (like frogs), but with simpler development patterns, faster growth, and transparency (similar to invertebrate models) .

• As vertebrates, zebrafish share significant sequence and functional homology with mammals, including humans, making findings potentially translatable to human disease processes .

• The zebrafish model permits whole-organism screening with complex phenotypes related to disease, offering advantages for studying systemic effects of ch25hl1.1 activity, particularly in neurovascular development .

• The developmental timeline of zebrafish is well-characterized, with brain angiogenesis and barrier function development occurring between 2-3 days post-fertilization, coinciding with when bleeding occurs in intracerebral hemorrhage models, making it valuable for studying ch25hl1.1's role in neurovascular development .

What are the available types of ch25hl1.1 antibodies and their basic properties?

Based on the available information, the ch25hl1.1 antibody from Cusabio (catalog number CSB-PA710054XA01DIL-0.2) has the following properties:

This antibody is designed specifically for zebrafish research and has been validated for specific applications in this model system .

What are the optimal protocols for validating ch25hl1.1 antibody specificity?

Validating antibody specificity is crucial, especially for zebrafish research where cross-reactivity can be problematic. Following a systematic validation approach:

Step 1: Understand the target

• Conduct a comprehensive literature review of ch25hl1.1 function and expression

• Note the biological relevance and expected subcellular localization of the protein

Step 2: Identify appropriate controls

• Use positive controls: Samples with known expression of ch25hl1.1

• Use negative controls: Samples where ch25hl1.1 is not expressed or knocked down

• Consider using 200μg of antigen provided with the antibody as a positive control

• Use the pre-immune serum provided as a negative control

Step 3: Multi-method validation

• Before immunohistochemistry, test the antibody in at least one other non-IHC method

• Western blot validation using zebrafish tissue lysates (not just recombinant protein) is recommended

• If targeting membrane proteins, consider flow cytometry against known positive cell lines

Step 4: Tier-appropriate validation
For ch25hl1.1 antibody, use Tier 3 validation (unknown antibody with limited literature evidence):

• Test multiple retrieval conditions to optimize staining

• Test antibody on putative positive and negative tissue

• Confirm correct cell type, compartment, and staining intensity

Step 5: Control of IHC experiments

• Include both positive and negative controls in each IHC run

• Use isotype-matched control antibody or omission of primary antibody to identify background staining

• For controls of polyclonal antibodies, use affinity-purified species-matched polyclonal or non-immune serum

How should ch25hl1.1 antibody be used for immunohistochemistry in zebrafish embryos?

Based on established protocols for zebrafish immunostaining:

• Fixation:

  • Fix zebrafish embryos with 4% formalin for 3 hours at room temperature or overnight at 4°C

  • Note that according to studies, some antibodies may lose immunoreactivity with prolonged fixation

• Washing and Blocking:

  • Wash with PBT (PBS with Tween)

  • Block with PBS containing 10% goat serum, 0.6% Triton and 1% DMSO for 1 hour at room temperature

• Primary Antibody Incubation:

  • Dilute purified antibody 1:1 in 1% goat serum, 0.6% Triton

  • Incubate overnight at 4°C

  • For ch25hl1.1 specifically, optimal dilutions should be determined by each laboratory for each application

• Secondary Antibody and Detection:

  • After washing in PBST, incubate with an appropriate secondary antibody (anti-rabbit-HRP)

  • For signal amplification, use a TSA (tyramide signal amplification) kit

  • For double labeling scenarios, consider using tagged antibodies detected with anti-tag antibodies

• Visualization:

  • For CH25H detection in zebrafish brain tissue, pay particular attention to regions around blood vessels and areas of potential hemorrhage

  • Document both positive staining and controls to demonstrate specificity

What are the important considerations for Western blot analysis using ch25hl1.1 antibody?

When using ch25hl1.1 antibody for Western blotting:

• Sample Preparation:

  • Use appropriate protein extraction methods that preserve the native state of ch25hl1.1

  • For membrane-associated proteins, consider using detergent-based lysis buffers

  • Test both non-reducing and reducing conditions, as some antibodies recognize conformational epitopes lost under reducing conditions

• Gel Selection and Protein Transfer:

  • Select appropriate percentage gels based on the expected size of ch25hl1.1 (check UniProt for theoretical molecular weight)

  • Transfer proteins to PVDF membranes, which often work better for hydrophobic proteins

• Blocking and Antibody Incubation:

  • Block in 2% BSA

  • Use ~10 μg/mL primary antibody for 1 hour at room temperature or overnight at 4°C

  • Include appropriate positive controls (such as the provided antigen) and negative controls

• Data Interpretation:

  • Be aware that proteins may not resolve as discrete bands but rather as smears due to glycoforms, particularly for secreted or membrane proteins

  • Confirm specificity by comparing with predicted molecular weight and control samples

  • Note any discrepancies between observed and expected band patterns for further investigation

How can ch25hl1.1 antibody be utilized to study neurovascular development and dysfunction in zebrafish?

Ch25hl1.1/CH25H has been implicated in neurovascular development and dysfunction, particularly in the context of viral-associated cerebral hemorrhage. The following methodological approach can be used:

• Developmental Timeline Analysis:

  • Use ch25hl1.1 antibody to track expression during critical windows of neurovascular development (2-3 dpf)

  • Compare expression patterns in normal development versus models of vascular stress or infection

• Colocalization Studies:

  • Perform double-staining with ch25hl1.1 antibody and endothelial markers

  • Analyze colocalization using confocal microscopy to determine proximity to vascular structures

  • Example approach: "To visualise microbleeds, stain slides with H&E protocol, then perform CH25H immunohistochemistry following antigen retrieval in Tris-EDTA (pH 9.0)"

• Viral Response Models:

  • Utilize spike-induced zebrafish ICH model to study ch25hl1.1 upregulation following viral stimulation

  • "Ch25h was transiently upregulated by spike injection before the increase in brain haemorrhage frequency"

  • Compare timing of ch25hl1.1 expression with onset of vascular phenotypes

• Quantitative Analysis:

  • Develop scoring systems for hemorrhage severity similar to those used in human samples:

  Bleeding Score	Characteristics
  0	Non-hemorrhagic samples
  1	Lower density of microbleeds
  2	Higher density of microbleeds, particularly medium-sized bleeds

  • Correlate ch25hl1.1-positive cell counts with bleeding scores to establish statistical relationships

• Drug Interaction Studies:

  • Combine ch25hl1.1 antibody staining with pharmacological interventions

  • Example approach from literature: "ATV incubation increased the expression of hmgcrb, a feedback response induced through the activation of SREBP2, and 25HC co-incubation inhibited this process"

  • Use ch25hl1.1 antibody to track expression changes following statin treatment or cholesterol supplementation

What are the methodological considerations for studying ch25hl1.1 in antiviral response pathways?

For investigating ch25hl1.1's role in antiviral responses:

• Temporal Expression Analysis:

  • Use the antibody to track ch25hl1.1 expression at different timepoints following viral stimulation

  • Compare expression kinetics with other antiviral markers and clinical outcomes

  • "Ch25h was transiently upregulated by spike injection before the increase in brain haemorrhage frequency"

• Cell-Type Specific Expression:

  • Perform double immunostaining with ch25hl1.1 antibody and cell-type specific markers

  • Determine which cell populations express ch25hl1.1 during antiviral responses

  • In human samples, "CH25H+ cells were apparent surrounding the granulomas typical of the infection"

• Functional Pathway Analysis:

  • Combine antibody staining with expression analysis of SREBP2 pathway components

  • Evaluate connections between ch25hl1.1 expression and cholesterol metabolism using the following methodological approach:

    • Measure accessible membrane cholesterol levels (using SLO permeability assays)

    • Evaluate expression of cholesterol synthesis genes (HMGCR, SQLE)

    • Analyze cholesterol efflux gene expression (ABCG1)

• Experimental Design for Mechanism Studies:

  • Study design for in vivo infection models that effectively assess the role of 25HC in antiviral responses while minimizing confounding factors

  • "Viral model considerations: Adult ICH may differ from developing brain and mild viral infection may only trigger adult ICH in combination with other risk factors"

How can ch25hl1.1 antibody be used in immunofluorescence applications for zebrafish research?

For immunofluorescence applications in zebrafish:

• Sample Preparation Optimization:

  • Fixation protocol: Determine optimal fixation time (3h at room temperature or overnight at 4°C)

  • Test multiple antigen retrieval conditions specific for ch25hl1.1 antibody

  • Consider tissue clearing techniques for deep tissue imaging

• Signal Amplification Strategies:

  • For weak signals, implement tyramide signal amplification (TSA)

  • Use recombinant antibody formats that enable convenient multiplex staining

  • Consider adding additional protein tags to recombinant antibodies to facilitate multiplex applications

• Advanced Imaging Approaches:

  • For studying ch25hl1.1 in the context of vascular structures:

    • Employ confocal microscopy for high-resolution 3D reconstruction

    • Consider light-sheet microscopy for whole-embryo imaging with reduced photobleaching

    • Implement live imaging for temporal dynamics in transgenic lines with fluorescent vascular markers

• Quantitative Analysis Methods:

  • Develop automated image analysis pipelines for:

    • Colocalization coefficients between ch25hl1.1 and other markers

    • Quantification of ch25hl1.1-positive cells relative to specific anatomical regions

    • Intensity measurement and comparison across experimental conditions

How can researchers overcome common challenges with ch25hl1.1 antibody staining in zebrafish?

Zebrafish-specific antibody challenges and solutions:

• Cross-Reactivity Issues:

  • Problem: Zebrafish glycans are highly immunogenic in mammals, leading to non-specific antibody binding

  • Solution: Use increased blocking concentrations (10% serum) and longer blocking times

  • Include additional washes with higher detergent concentrations to reduce background

• Fixation Sensitivity:

  • Problem: Some antibody epitopes are sensitive to formalin fixation

  • Solution: Test multiple fixation conditions and times

  • Quantitatively assess the rate at which immunoreactivity is lost upon formalin treatment using ELISA

• Signal Strength Optimization:

  • Problem: Weak signal from low-abundance targets

  • Solution: Implement signal amplification methods like TSA

  • Consider using rabbit monoclonal antibodies when available, as they typically offer higher sensitivity than polyclonals

• Validation in Multiple Applications:

  • Problem: Antibody may work in one application but not another

  • Solution: Systematically validate in multiple applications (WB, IHC, IF)

  • "Compare sample data determined on 2 independent assays runs" to ensure reproducibility

• Specific ch25hl1.1 Challenges:

  • Develop a targeted optimization strategy focusing on factors most relevant to membrane-associated proteins

  • Consider detergent concentration, fixation time, and antigen retrieval method optimization specific to ch25hl1.1

What quality control measures should be implemented when using ch25hl1.1 antibody in experimental workflows?

Comprehensive quality control measures:

• Reproducibility Testing:

  • Run independent experiments on different days with different antibody lots

  • Calculate coefficient of variation (%CV) between runs

  • Aim for %CV values <25% as acceptable reproducibility criterion

• Specificity Controls:

  • Use provided positive control (200μg antigen) and negative control (pre-immune serum)

  • Include additional specificity controls:

    • Omission of primary antibody

    • Isotype-matched controls

    • Blocking peptide competition assay if available

• Batch Consistency Monitoring:

  • Maintain reference samples to test each new antibody lot

  • Document lot-to-lot variation with standardized control samples

  • Consider preparing a cell line microarray (CMA) with known positive and negative control cells

• Statistical Validation:

  • For quantitative applications, determine limit of detection (LoD)

  • Establish standard curves using recombinant protein controls

  • Document linear range of detection for each application

• Documentation Requirements:

  • Record complete antibody metadata: catalog number, lot, dilution, incubation conditions

  • Follow MISFISHIE guidelines for reporting antibody-based experiments

  • Include appropriate control material in publications either within main figures or as supplementary material

How can ch25hl1.1 antibody specificity be definitively confirmed through advanced techniques?

Advanced validation approaches to definitively confirm ch25hl1.1 antibody specificity:

• Genetic Knockout/Knockdown Validation:

  • Generate ch25hl1.1 knockout or knockdown zebrafish models using CRISPR/Cas9 or morpholinos

  • Compare antibody staining between wild-type and knockout/knockdown animals

  • Absence of staining in knockout/knockdown models provides strong evidence of specificity

• Mass Spectrometry Confirmation:

  • Use the antibody for immunoprecipitation experiments

  • Analyze the precipitated proteins by mass spectrometry

  • Confirm the identity of the target protein to verify antibody specificity

• Recombinant Expression Systems:

  • Express tagged versions of ch25hl1.1 in heterologous systems

  • Compare antibody staining with anti-tag antibodies to confirm colocalization

  • This approach works particularly well for antibodies against proteins with low endogenous expression

• Multi-Antibody Validation ("Sibling Antibodies"):

  • Use multiple antibodies raised against different epitopes of the same protein

  • "The use of 'sibling antibodies,' where multiple antibodies are prepared to the same target, can add confidence in the data quality"

  • Consistent staining patterns across multiple antibodies increases confidence in specificity

• Cross-Species Validation:

  • Test reactivity against ch25h homologs from different species

  • Compare observed staining patterns with predicted evolutionary conservation

  • This approach can help distinguish specific from non-specific binding

How can ch25hl1.1 antibody contribute to research on cholesterol metabolism in neurovascular disease?

The ch25hl1.1 antibody can be used to investigate several aspects of cholesterol metabolism in neurovascular disease:

• Metabolism Pathway Analysis:

  • Use ch25hl1.1 antibody to track expression changes in response to metabolic perturbations

  • Combine with markers of cholesterol synthesis and transport:

    • HMGCR (rate-limiting enzyme in cholesterol synthesis)

    • SREBP2 (master transcriptional regulator)

    • ABCG1 (cholesterol efflux transporter)

• Disease Model Characterization:

  • Apply the antibody in zebrafish models of cerebrovascular disorders

  • Research approach: "In SARS-CoV-2-spike injected zebrafish larvae and human SARS-CoV-2-associated developmental brain haemorrhages, we identified an upregulation of CH25H"

  • Compare ch25hl1.1 expression patterns in models with differing severity of vascular phenotypes

• Pharmacological Intervention Studies:

  • Monitor ch25hl1.1 expression changes following treatment with:

    • Statins (HMGCR inhibitors)

    • Cholesterol supplementation

    • LXR agonists (regulators of cholesterol efflux)

  • Research finding: "ATV and 25HC co-treatment significantly increased permeability in comparison to 25HC alone"

• Translational Research Approaches:

  • Compare zebrafish findings with human patient samples

  • Experimental design: "In vitro 25HC-induced dysfunction was also rescued by cholesterol supplementation"

  • Develop targeted therapeutic strategies based on ch25hl1.1 pathway modulation

What are the methodological considerations for using ch25hl1.1 antibody in developmental biology research?

For developmental biology applications:

• Temporal Expression Mapping:

  • Use ch25hl1.1 antibody to document expression patterns across developmental stages

  • Focus on critical windows of neurovascular development (2-3 dpf in zebrafish)

  • Compare with established developmental markers to identify co-expression patterns

• Lineage Tracing Integration:

  • Combine ch25hl1.1 antibody staining with transgenic lineage markers

  • Methods for sample preparation: "Zebrafish embryos were fixed with 4% formalin for 3h at room temperature or 4°C overnight, washed with PBT, and blocked (PBS, 10% goat serum, 0.6% Triton and 1% DMSO)"

  • Analyze cell fate decisions in ch25hl1.1-expressing populations

• Morphogenetic Process Analysis:

  • Evaluate ch25hl1.1 expression during key morphogenetic events

  • Particular focus on vascular development processes:

    • Angiogenesis

    • Blood-brain barrier formation

    • Vessel stabilization

• Functional Perturbation Studies:

  • Combine antibody staining with:

    • Genetic knockdown/knockout approaches

    • Small molecule inhibitors

    • Targeted protein degradation

  • Compare expression patterns before and after perturbation to understand functional significance

How can researchers design experiments to investigate ch25hl1.1's role in infection-induced vascular pathology?

Experimental design considerations for studying ch25hl1.1 in infection-induced vascular pathology:

• Model Selection and Characterization:

  • Choose appropriate models that recapitulate infection-associated vascular pathology:

    • "SARS-CoV-2-spike-induced zebrafish ICH model"

    • "Foetal human SARS-CoV-2-associated cortical tissue containing microbleeds"

  • Define clear phenotypic readouts and scoring systems

• Mechanistic Dissection Approach:

  • Use ch25hl1.1 antibody in combination with:

    • Time-course studies to determine temporal relationship between expression and pathology

    • Pharmacological interventions targeting specific pathway components

    • Genetic manipulation of upstream and downstream factors

  • Research finding: "25HC and pharmacological inhibition of HMGCR by atorvastatin interacted to exacerbate brain bleeding in zebrafish larvae"

• Translational Research Design:

  • Compare findings between zebrafish models and human samples

  • Develop standardized protocols for quantifying ch25hl1.1 expression:

    • "CH25H levels were evaluated by immunohistochemistry, observing a lack of CH25H+ cells in non-haemorrhagic samples and variable numbers of CH25H+ cells in haemorrhagic samples"

  • Correlate expression patterns with clinical outcomes

• Combined In Vivo and In Vitro Approaches:

  • Use ch25hl1.1 antibody across complementary experimental systems:

    • In vivo zebrafish models for systemic effects

    • In vitro brain endothelial cell cultures for mechanistic studies

  • Translational finding: "25HC-induced dysfunction was also rescued by cholesterol supplementation. These results demonstrate that the antiviral factor 25HC can dysregulate brain endothelial function by remodelling cholesterol metabolism"

What emerging technologies could enhance the utility of ch25hl1.1 antibodies in zebrafish research?

Several emerging technologies could significantly enhance ch25hl1.1 antibody applications:

• Advanced Recombinant Antibody Engineering:

  • Development of recombinant ch25hl1.1 antibodies with customized properties:

    • Site-specific conjugation for controlled labeling

    • Smaller antibody formats (nanobodies, single-chain fragments)

    • Bifunctional antibodies for multiplexed detection

  • Research advantage: "Recombinant antibody production allows researchers more control over the antigen"

• Spatial Transcriptomics Integration:

  • Combine ch25hl1.1 antibody staining with spatial transcriptomics:

    • Correlate protein expression with local transcriptional profiles

    • Identify regulatory networks controlling ch25hl1.1 expression

    • Map cell-cell communication networks in ch25hl1.1-expressing regions

• Advanced Imaging Technologies:

  • Implement emerging imaging approaches:

    • Expansion microscopy for super-resolution imaging

    • Light-sheet microscopy for whole-organism imaging

    • Intravital microscopy for real-time dynamics

  • These approaches allow visualization of ch25hl1.1 expression in relation to fine vascular structures

• Computational Analysis Integration:

  • Apply machine learning approaches to:

    • Automatically quantify ch25hl1.1-positive cells across tissues

    • Classify expression patterns across developmental stages

    • Correlate expression with phenotypic outcomes

How can computational modeling be integrated with ch25hl1.1 antibody data to advance understanding of its function?

Computational approaches that integrate ch25hl1.1 antibody data:

• Systems Biology Modeling:

  • Integrate ch25hl1.1 antibody expression data into pathway models:

    • Map interactions between cholesterol metabolism and innate immunity

    • Predict system-level responses to perturbations

    • Identify feedback mechanisms and control points

  • Relevant pathway interactions: "25HC modulated cholesterol metabolism at a transcriptional level and by reducing accessible cholesterol levels in the plasma membrane"

• Machine Learning Classification:

  • Apply supervised learning to:

    • Classify cellular phenotypes based on ch25hl1.1 expression patterns

    • Identify predictive features of vascular dysfunction

    • Develop automated scoring systems for hemorrhage severity

  • Example approach: "Measuring the number and size of microbleeds, we classified the cortical samples by a bleeding score"

• Image Analysis Automation:

  • Develop computational pipelines for:

    • High-throughput quantification of ch25hl1.1 expression

    • 3D reconstruction of expression patterns in relation to vascular networks

    • Temporal dynamics analysis across developmental stages

  • This addresses the challenge of analyzing large datasets generated by whole-organism imaging

• Multi-omics Data Integration:

  • Combine antibody-based protein expression data with:

    • Transcriptomics (RNA-seq)

    • Metabolomics (especially cholesterol metabolites)

    • Epigenomics (regulatory landscape)

  • Generate comprehensive models of how ch25hl1.1 functions within broader biological networks

What are the most pressing research questions regarding ch25hl1.1 function that antibody-based approaches could help address?

Critical research questions that could be addressed using ch25hl1.1 antibodies:

• Cell-Type Specific Roles:

  • Which cell types express ch25hl1.1 during different physiological and pathological conditions?

  • How does cell-type specific expression contribute to tissue-specific responses?

  • Methodological approach: "Use double-staining with ch25hl1.1 antibody and cell-type specific markers"

• Temporal Dynamics of Expression:

  • What is the precise timing of ch25hl1.1 upregulation relative to infection or injury?

  • How does expression change throughout development and aging?

  • Research finding: "Ch25h was transiently upregulated by spike injection before the increase in brain haemorrhage frequency"

• Subcellular Localization and Trafficking:

  • Where within cells is ch25hl1.1 localized under different conditions?

  • How does subcellular distribution affect function?

  • Approach: Use high-resolution microscopy with ch25hl1.1 antibody and organelle markers

• Functional Interactions:

  • What proteins physically interact with ch25hl1.1?

  • How do these interactions change during antiviral responses?

  • Methods: Combine immunoprecipitation with mass spectrometry to identify interaction partners

• Therapeutic Targeting Potential:

  • Could modulation of ch25hl1.1 activity protect against infection-associated vascular pathology?

  • What are the potential off-target effects of such interventions?

  • Finding: "The CH25H/25HC pathway may be a relevant factor in infection-triggered brain EC dysfunction"