ALOXE3 Antibody

What is ALOXE3 and what is its biological significance?

ALOXE3, also known as epidermis-type lipoxygenase 3 (e-LOX-3), is a non-heme iron-containing lipoxygenase with unique biochemical properties. Unlike typical lipoxygenases, ALOXE3 displays prominent hydroperoxide isomerase activity while exhibiting reduced lipoxygenase activity . This enzyme catalyzes the isomerization of hydroperoxides derived from arachidonic and linoleic acid (primarily those generated by ALOX12B) into hepoxilin-type epoxyalcohols and ketones .

ALOXE3 has multiple biological functions across different tissues:

• Skin barrier formation: In skin, ALOXE3 acts downstream of ALOX12B on the linoleate moiety of esterified omega-hydroxyacyl-sphingosine (EOS) ceramides to produce epoxy-ketone derivatives. This represents a crucial step in conjugating omega-hydroxyceramide to membrane proteins, which is essential for establishing the skin barrier against water loss .

• Neural function: ALOXE3 expression in the brain suggests roles in modulating neural excitability. Its spatiotemporal expression pattern implies involvement in brain development and seizure susceptibility .

• Metabolic regulation: Emerging evidence indicates ALOXE3 functions as a hepatic fasting-responsive lipoxygenase, potentially affecting metabolic processes .

What types of anti-ALOXE3 antibodies are available for research purposes?

Based on the search results, several types of anti-ALOXE3 antibodies are available for research:

• Polyclonal antibodies: Rabbit polyclonal antibodies against human ALOXE3 are available, including those targeting recombinant fragment proteins within human hydroperoxide isomerase ALOXE3 .

• Application-validated antibodies: Antibodies validated for specific applications including:

  • Western blotting (WB)

  • Immunohistochemistry on paraffin-embedded sections (IHC-P)

  • Immunocytochemistry/immunofluorescence (ICC-IF)

• Species-specific antibodies: The search results mention antibodies that react with human samples, though antibodies against mouse ALOXE3 are also referenced in the research studies .

The choice of antibody depends on the specific research application, target species, and experimental conditions. When selecting an ALOXE3 antibody, researchers should consider validation data, cross-reactivity profile, and appropriate positive controls for their experimental system.

How should researchers validate the specificity of ALOXE3 antibodies?

Validating antibody specificity is crucial for generating reliable research data. For ALOXE3 antibodies, multiple validation approaches should be employed:

Western blot validation: The specificity of ALOXE3 antibodies can be confirmed by western blot analysis. As described in the research, strong immunoreactive bands should be observed at the predicted molecular weight (~80 kD) in relevant tissues such as hippocampus and temporal cortex . Researchers should verify that their antibody recognizes a single band of the expected size.

Negative controls: When performing immunohistochemistry or immunofluorescence, include negative controls using only secondary antibodies to assess background staining. As noted in the search results: "For immunohistochemistry assay, unwanted background was not observed in the negative control only incubated with the secondary antibody" .

Comparative analysis: Compare antibody performance against known expression patterns. For example, ALOXE3 shows age-dependent expression in mouse hippocampus and temporal cortex, with increasing levels from postnatal day 1 (P1) to P30 .

Cross-validation with different antibodies: When available, use multiple antibodies targeting different epitopes of ALOXE3. The search results mention two different ALOXE3 antibodies:

• Anti-ALOXE3 (1:200, Cat#: ab118470, Abcam)

• Anti-ALOXE3 (1:200, Cat#: PA5-23953, Thermo Fisher Scientific)

Genetic validation: When possible, use tissue from knockout models or cells with gene silencing as controls for antibody specificity.

What is the spatiotemporal expression pattern of ALOXE3 in the brain and its implications for neurological research?

The spatiotemporal expression pattern of ALOXE3 in the brain reveals important insights for neurological research:

Temporal expression pattern:

• ALOXE3 expression increases progressively with age in the mouse brain

• Aloxe3 mRNA levels significantly increase from postnatal day 15 (P15) through P60

• ALOXE3 protein is nearly undetectable during the neonatal period (P1)

• Protein expression becomes detectable by the end of the first week (P7)

• Levels continue to increase until P30, then stabilize between P30 and P60

Spatial distribution:

• Strongest expression occurs in the hippocampal formation, particularly in the CA1 subregion and mossy fiber area

• Moderate expression in CA3 region and dentate gyrus

• Prominent staining in hippocampal pyramidal cell layer, soma, and apical dendrites

• High staining in stratum oriens and stratum radiatum neuropil

• In temporal cortex, expression is primarily in somatosensory cortex, especially within layers I–IV/V and V

• Punctate appearance in layers I-III with scattered positive cells in layers IV-V

Subcellular localization:

• ALOXE3 distribution is restricted to neurites of function-specific subregions

• Present in mossy fibers connecting hilus and CA3 neurons

• Found in termini of Schaffer collateral projections

• Expressed in layers III and IV of somatosensory cortex

Research implications:
This pattern suggests ALOXE3 plays critical roles in:

• Neural development during postnatal brain maturation

• Modulation of neural excitability in specific circuits

• Regulation of seizure susceptibility through arachidonic acid metabolism

Researchers investigating neurological disorders, particularly those involving excitability imbalances like epilepsy, should consider ALOXE3 as a potential regulator of neural function through its effects on arachidonic acid metabolism and hepoxilin production.

What methodological approaches are most effective for studying ALOXE3 function in seizure models?

Based on the search results, several methodological approaches have proven effective for studying ALOXE3 function in seizure models:

Gene manipulation via viral vectors:

• AAV-mediated overexpression of ALOXE3 through intrahippocampal injection (AAV2-ALOXE3)

• Verification of overexpression using western blot analysis

• This approach successfully restored elevated AA levels in seizure models and reduced seizure susceptibility

Biochemical measurement of arachidonic acid (AA) levels:

• Quantifying AA concentration in hippocampal tissue following status epilepticus (SE)

• Comparing AA levels between control and ALOXE3-overexpressing animals

• Statistical analysis using Kruskal Wallis followed by Dunn's post hoc test when data doesn't meet homogeneity of variance assumptions

Behavioral assessment of seizure parameters:

• Measuring latency to myoclonic switch

• Recording onset of clonic convulsions

• Monitoring tonic hindlimb extensions

• Calculating mortality rates using Chi-square test

• Determining latency to death (Kruskal Wallis followed by Dunn's post hoc test)

Immunohistochemical analysis:

• Using anti-ALOXE3 antibody (1:200, Cat#: ab118470, Abcam)

• Processing with biotinylated secondary antibodies

• Visualizing with avidin–biotin-peroxidase complex

• Staining with diaminobenzidine and H₂O₂

• Observing using digital microscopy

Co-localization studies via immunofluorescence:

• Double-labeling with ALOXE3 and neuronal markers (NeuN)

• Using synaptic markers (ZnT3, Synapsin1, VGLUT1, VGAT)

• Applying appropriate secondary antibodies (goat anti-rabbit IgG Cy3-conjugated and anti-mouse IgG FITC-conjugated)

• Analyzing using confocal microscopy

These methods, when combined, provide comprehensive insights into both the molecular function of ALOXE3 in regulating neural excitability and its potential therapeutic relevance in seizure disorders.

What are the optimal conditions for using ALOXE3 antibodies in different experimental applications?

Optimal conditions for using ALOXE3 antibodies vary by application. Based on the search results, here are evidence-based recommendations:

Western Blot (WB):

• Expected molecular weight: ~80 kD for ALOXE3

• Antibody dilution: Not specifically stated in search results, but typically 1:500-1:2000 for most primary antibodies

• Sample preparation: Brain tissue homogenates should be prepared with protease inhibitors

• Confirmation: The specificity of anti-ALOXE3 in brain tissue shows strong immunoreactive bands at the predicted location (~80 kD) in both hippocampus and temporal cortex

Immunohistochemistry (IHC):

• Sample preparation: Paraformaldehyde-fixed, paraffin-embedded or frozen sections

• Antigen retrieval: Treatment with 0.3% H₂O₂ for 30 minutes

• Blocking: 10% normal donkey serum for 1 hour at room temperature

• Primary antibody: Anti-ALOXE3 (1:200, Cat#: ab118470, Abcam), incubated overnight at 4°C

• Secondary antibody: Biotinylated secondary immunoglobulin G antibody at room temperature for 2 hours

• Detection: Avidin–biotin-peroxidase complex for 30 minutes, followed by 0.05% diaminobenzidine and 0.01% H₂O₂

• Visualization: Digital microscopy (Leica DMI4000 B)

Immunofluorescence (IF):

• Primary antibody: Anti-ALOXE3 (1:200, Cat#: PA5-23953, Thermo Fisher Scientific)

• Co-staining options:

  • Anti-NeuN (1:4000, Cat#: PA5-78499, Millipore)

  • Anti–ZnT3 (1:500, Cat#: 197011, Synaptic Systems)

  • Anti-Synapsin1 (1:500, Cat#: 106011, Synaptic Systems)

  • Anti-VGLUT1 (1:500, Cat#: 135304, Synaptic Systems)

  • Anti-VGAT (1:500, Cat#: 131004, Synaptic Systems)

• Secondary antibodies:

  • Goat anti-rabbit IgG Cy3-conjugated (1:100, Cat#: AP132C, Millipore)

  • Anti-mouse IgG FITC-conjugated (1:100, Cat#: AP308F, Millipore)

• Visualization: Confocal microscopy (Leica SP8)

Important considerations:

• Negative controls: Always include sections incubated with only secondary antibody

• Antibody validation: Confirm specificity via western blot

• Age-dependent expression: Consider the developmental stage when studying ALOXE3 (expression increases with age from P1 to P30)

• Tissue-specific expression: Strongest in hippocampus, temporal cortex, and striatum

How does ALOXE3 function differ between skin and neural tissues, and what implications does this have for antibody-based research?

ALOXE3 exhibits distinct functions in skin versus neural tissues, which has important implications for antibody-based research:

Functional differences:

In skin:

• ALOXE3 acts downstream of ALOX12B on the linoleate moiety of esterified omega-hydroxyacyl-sphingosine (EOS) ceramides

• Produces epoxy-ketone derivatives crucial for conjugating omega-hydroxyceramide to membrane proteins

• Plays a critical role in synthesizing the corneocytes lipid envelope

• Establishes the skin barrier to prevent water loss

• May have signaling functions in barrier formation through hepoxilin metabolites

In neural tissue:

• Involved in arachidonic acid (AA) metabolism in the brain

• Acts as a regulator of neural excitability

• Controls brain development and seizure susceptibility

• Overexpression reduces seizure susceptibility in experimental models

• Restores elevated AA levels induced by status epilepticus

Implications for antibody-based research:

• Target epitope selection: Antibodies targeting different epitopes may be needed to study tissue-specific functions. Researchers should select antibodies that recognize epitopes relevant to the tissue-specific conformation or post-translational modifications.

• Experimental controls: Different positive controls should be used:

  • For skin studies: Epidermal tissue samples with known ALOXE3 expression

  • For neural studies: Adult hippocampus, particularly CA1 and mossy fiber regions

• Developmental timing: When studying brain tissue, the age of the specimen is critical:

  • ALOXE3 is nearly undetectable at P1

  • Expression increases gradually through P30

  • Stabilizes between P30-P60
    Therefore, timing of sample collection is crucial for neural studies.

• Co-localization partners:

  • In skin: Consider co-staining with epidermal markers

  • In brain: Consider co-staining with neuronal markers (NeuN) or synaptic markers (ZnT3, Synapsin1, VGLUT1, VGAT) depending on the research question

• Subcellular localization:

  • In skin: Membrane-associated and cytoplasmic

  • In brain: Neurite-specific distribution in function-specific subregions (mossy fibers, Schaffer collateral termini)

• Functional readouts:

  • For skin studies: Barrier function tests

  • For neural studies: AA level measurement, seizure susceptibility parameters

Understanding these tissue-specific differences helps researchers design appropriate antibody validation strategies and experimental approaches for their specific tissue of interest.

What technical challenges exist in studying ALOXE3 expression and function, and how can they be overcome?

Several technical challenges exist in studying ALOXE3, with evidence-based solutions from the search results:

Developmental regulation challenges:

Challenge: ALOXE3 expression is developmentally regulated, with expression nearly undetectable in early development (P1) and increasing significantly through P30 .

Solutions:

• Use age-appropriate tissue samples

• Implement more sensitive detection methods for early developmental stages

• Normalize expression data to appropriate housekeeping genes

• Consider using amplification steps in immunohistochemistry for early developmental stages

Antibody specificity issues:

Challenge: Ensuring antibody specificity, especially in tissues with potentially low expression.

Solutions:

• Validate antibodies by western blot to confirm the expected ~80 kD band

• Include negative controls (secondary antibody only) in all experiments

• Use multiple antibodies targeting different epitopes when possible

• Consider using tissues from knockout models as negative controls

• Pre-absorb antibodies with the immunizing peptide to confirm specificity

Complex spatiotemporal expression pattern:

Challenge: ALOXE3 shows region-specific expression in the brain, requiring precise anatomical localization.

Solutions:

• Use stereotaxic coordinates for precise targeting in mouse brain studies

• Employ double-labeling with region-specific markers (e.g., NeuN, ZnT3)

• Utilize confocal microscopy for detailed subcellular localization

• Consider micro-dissection techniques to isolate specific brain regions

Functional redundancy with other lipoxygenases:

Challenge: Distinguishing ALOXE3-specific functions from those of other lipoxygenases.

Solutions:

• Use targeted gene manipulation (AAV-mediated overexpression)

• Measure specific metabolites (e.g., hepoxilins) rather than just substrate levels

• Conduct parallel studies with inhibitors of different lipoxygenases

• Perform rescue experiments in systems where ALOXE3 is downregulated

Statistical analysis of variable biological responses:

Challenge: Biological responses like seizure susceptibility can show high variability.

Solutions:

• Use appropriate statistical tests based on data distribution (e.g., Kruskal Wallis followed by Dunn's post hoc test for non-homogenous variance)

• Increase sample sizes to account for variability

• Report effect sizes (e.g., partial η²) to communicate practical significance

• Use mixed-effects models to account for repeated measures and covariates

Measuring relevant metabolic products:

Challenge: Quantifying AA and hepoxilins accurately.

Solutions:

• Standardize tissue collection and processing procedures

• Use mass spectrometry-based approaches for metabolite quantification

• Include appropriate internal standards

• Consider using stable isotope labeling to track metabolic flux

By addressing these technical challenges with the suggested solutions, researchers can design more robust studies of ALOXE3 expression and function, leading to more reliable and reproducible results.