HEPACAM Antibody, FITC conjugated

What is HEPACAM and what cellular functions does it regulate?

HEPACAM, also known as glialCAM or Hepatocyte Cell Adhesion Molecule, is a glycoprotein involved in regulating cell motility and cell-matrix interactions. It plays a significant role in cell adhesion processes and may inhibit cell growth through suppression of cell proliferation . In glial cells, HEPACAM associates with and targets CLCN2 (chloride channel protein 2) at astrocytic processes and myelinated fiber tracts, potentially regulating transcellular chloride flux involved in neuron excitability . The protein is expressed at notable levels in brain tissue, particularly in the cerebrum, where it demonstrates characteristic cytoplasmic staining patterns when detected via immunohistochemistry . HEPACAM is a glycoprotein with a molecular weight ranging from approximately 46-72 kDa, though it can be detected as a 46 kDa band after treatment with protein deglycosylation enzymes, indicating significant post-translational modifications .

How does a FITC-conjugated HEPACAM antibody differ from unconjugated versions?

FITC-conjugated HEPACAM antibodies contain the fluorescein isothiocyanate fluorophore covalently linked to the antibody molecule, enabling direct visualization without secondary detection reagents. Unlike unconjugated primary antibodies that require labeled secondary antibodies for detection, FITC-conjugated antibodies provide immediate fluorescent signaling upon binding to the target antigen. This direct labeling approach eliminates potential cross-reactivity issues associated with secondary antibodies and simplifies multi-color staining protocols. In research applications, FITC-conjugated HEPACAM antibodies have been effectively utilized for fluorescence-based cell sorting, where cells can be gated based on the presence of FITC-conjugated HepaCAM antibodies . This direct labeling strategy is particularly advantageous in flow cytometry applications, allowing researchers to distinguish between HepaCAM-positive and HepaCAM-negative cell populations with high specificity and without additional staining steps.

What are the validated applications for HEPACAM antibodies in brain tissue research?

HEPACAM antibodies have been validated for multiple research applications involving brain tissue analysis. Western blot analysis has confirmed specific detection of HEPACAM in human brain (motor cortex and hippocampus) tissue lysates, with the protein appearing as bands between 45-70 kDa under reducing conditions . Immunohistochemical analysis of paraffin-embedded human and mouse cerebrum tissue has demonstrated reliable cytoplasmic staining patterns when using specific anti-HEPACAM antibodies . In immunofluorescence applications, HEPACAM antibodies have enabled the visualization of the protein at cell-cell contacts, particularly in studies investigating its co-localization with connexin 43 . Additionally, HEPACAM antibodies have proven effective in immunopanning protocols for isolating astrocyte lineage cells from human cerebral cortical spheroids (hCS), allowing for the enrichment and subsequent characterization of these cells through downstream applications like RNA-seq transcriptional profiling .

What are the typical expression patterns of HEPACAM in neural tissues?

HEPACAM exhibits distinct expression patterns in neural tissues, with significant presence in both human and mouse cerebrum. Immunohistochemical analyses reveal primarily cytoplasmic staining in cerebral tissues . In functional contexts, HEPACAM is notably expressed at cell-cell contacts, where it co-localizes with connexin 43, suggesting its involvement in cell-cell communication mechanisms . Western blot analyses of mouse and rat brain tissue lysates consistently detect HEPACAM at approximately 46 kDa, though the glycosylated form can appear at higher molecular weights . HEPACAM expression in the human dorsal pallium follows developmental progression, with immunopanning using anti-HEPACAM antibodies becoming increasingly efficient after 100 days of differentiation in vitro, indicating a delayed onset of astrocyte generation that typically begins after the substantive portion of neurogenesis is complete . This temporal expression pattern aligns with established developmental trajectories in which astrocyte generation follows neuronal production in the mammalian brain.

How can HEPACAM antibody, FITC conjugated be optimized for flow cytometry-based cell sorting?

Optimizing FITC-conjugated HEPACAM antibodies for flow cytometry requires careful consideration of several methodological factors. For effective cell sorting applications, researchers should first determine the optimal antibody concentration through titration experiments, typically starting with the manufacturer's recommended dilution and testing 2-3 concentrations above and below this value to maximize signal-to-noise ratio. When working with human cerebral cortical spheroids (hCS), single-cell suspensions must be carefully prepared, with researchers reporting successful yields of approximately 500,000–1,000,000 live cells per hCS . Incubation time and temperature also require optimization, with most protocols recommending 20-30 minutes at 4°C to minimize antibody internalization while maintaining cell viability.

For multi-parameter analysis, compensation controls must be established to account for spectral overlap between FITC and other fluorophores. Since HEPACAM is a cell surface glycoprotein, researchers should avoid harsh fixation methods that might denature surface epitopes prior to antibody labeling. In studies isolating astrocyte lineage cells, gating strategies involving FITC-conjugated HEPACAM antibodies have proven effective, with increasing efficiency observed after 100 days of differentiation in vitro, corresponding to the delayed onset of astrocyte generation in human dorsal pallium development . This temporal consideration is crucial when designing experiments focused on developmental progression of neural cell populations.

What is the relationship between HEPACAM and connexin 43, and how can this be studied using fluorescent antibodies?

HEPACAM demonstrates a significant functional relationship with connexin 43, a gap junction protein crucial for intercellular communication. Co-immunoprecipitation experiments have confirmed that HEPACAM physically associates with connexin 43 in U373 MG cells stably transfected with wild-type HEPACAM . This association has functional consequences, as expression of wild-type HEPACAM significantly increases connexin 43 protein levels, with quantification showing approximately two-fold higher expression compared to control cells .

For studying this relationship using fluorescent antibodies, dual immunofluorescence staining provides valuable insights. Researchers have successfully visualized the co-localization of HEPACAM and connexin 43 at cell-cell contacts using antibodies against the HEPACAM cytoplasmic domain (green fluorescence) and connexin 43 (red fluorescence), with co-localization appearing as yellow fluorescence in merged images . Confocal microscopy with a 60× objective allows detailed visualization of this interaction, particularly at cell-cell contact sites. Interestingly, treatment with antibodies against the HEPACAM extracellular domain disrupts this association and causes downregulation of connexin 43 expression, providing a methodological approach to studying the functional consequences of this interaction . For quantitative assessment, Western blot analysis of connexin 43 levels normalized to loading controls such as GAPDH, followed by densitometric analysis using software like ImageJ, enables statistical evaluation of the effects of HEPACAM on connexin 43 expression across experimental conditions.

How can HEPACAM antibodies be used to study the maturation of human astrocytes in 3D cerebral cortical spheroids?

HEPACAM antibodies represent a powerful tool for studying astrocyte maturation in 3D cerebral cortical spheroids (hCS) through immunopanning techniques. Researchers have adapted immunopanning protocols originally developed for primary human fetal and adult brain tissue to isolate astrocyte lineage cells from hCS . This approach involves passing a single-cell suspension of dissociated hCS over cell-culture plates coated with antibodies against cell-type specific antigens, including anti-HEPACAM antibodies to bind astrocytes and anti-Thy-1 antibodies to isolate neurons .

The effectiveness of HEPACAM-based immunopanning for astrocyte isolation increases with differentiation time, becoming significantly more efficient after 100 days of in vitro differentiation, which correlates with the developmental timeline of astrocyte generation following neurogenesis in the human dorsal pallium . This temporal pattern allows researchers to track astrocyte maturation by analyzing HEPACAM-expressing cells at different developmental timepoints. Following isolation, HEPACAM-positive cells can be further characterized using RNA-seq transcriptional profiling to verify expression of established astrocyte markers such as Aquaporin-4, distinguishing mature astrocytes from neurons and radial glia .

For fluorescence-based studies, FITC-conjugated HEPACAM antibodies enable direct visualization and quantification of astrocyte populations within heterogeneous cultures, particularly when combined with markers like GFAP, though it's important to note that GFAP expression can be variable in mature astrocytes and is also present in radial glia . This multiparameter approach provides a more comprehensive assessment of astrocyte maturation states than reliance on any single marker.

What controls should be included when using FITC-conjugated HEPACAM antibodies for immunofluorescence?

Implementing appropriate controls is essential when using FITC-conjugated HEPACAM antibodies for immunofluorescence applications. First, isotype controls matching the HEPACAM antibody's host species and immunoglobulin class are crucial to assess potential non-specific binding. In published studies, mouse IgG1 has been used as a negative control for mouse monoclonal anti-HEPACAM antibodies . Second, negative tissue controls utilizing samples known not to express HEPACAM should be included; LNCaP cell lines have been identified as appropriate negative controls in certain contexts .

For multicolor imaging applications, single-stained controls for each fluorophore are necessary to establish proper compensation settings and account for spectral overlap. Additionally, secondary-only controls (using fluorescently labeled secondary antibodies without primary antibodies) help evaluate background fluorescence levels, as demonstrated in immunohistochemical protocols using LeicaDS9800 (Bond Polymer Refine Detection) systems . Positive controls are equally important and should include tissues or cell lines with verified HEPACAM expression; human and mouse cerebrum tissues have been validated for this purpose .

When examining co-localization with other proteins like connexin 43, parallel samples treated with blocking antibodies against the HEPACAM extracellular domain provide valuable functional controls, as this treatment has been shown to prevent HEPACAM-connexin 43 association at cell-cell contacts . These comprehensive control strategies ensure reliable and interpretable results when using FITC-conjugated HEPACAM antibodies for immunofluorescence studies.

How should sample preparation be optimized for detecting HEPACAM in brain tissue specimens?

Optimal detection of HEPACAM in brain tissue specimens requires careful consideration of fixation, antigen retrieval, and processing methods. For paraffin-embedded tissue sections, heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes has proven effective in preserving HEPACAM epitope accessibility . When working with fresh-frozen sections, a brief 10-minute fixation in 4% paraformaldehyde followed by careful permeabilization with 0.1% Triton X-100 typically yields good results while maintaining structural integrity.

For Western blot applications involving brain tissue lysates, sample preparation should account for HEPACAM's glycoprotein nature. Notably, HEPACAM appears as a diffuse band between 46-72 kDa in its native glycosylated state but reduces to approximately 46 kDa after treatment with deglycosylation enzymes . Samples should generally remain unboiled to preserve epitope structure, with blocking in 5% non-fat dry milk in TBST providing optimal background reduction .

When preparing single-cell suspensions from human cerebral spheroids for immunopanning or flow cytometry, gentle dissociation methods are essential to preserve cell surface epitopes. For immunopanning applications targeting HEPACAM-positive astrocytes, researchers have successfully obtained 500,000-1,000,000 viable cells per cerebral spheroid . Following dissociation, maintaining cells at 4°C and minimizing processing time before antibody labeling helps preserve surface antigen integrity. These methodological considerations significantly impact the quality and reliability of HEPACAM detection in various experimental contexts.

What factors influence the variability in HEPACAM detection using antibody-based methods?

Several critical factors contribute to variability in HEPACAM detection across antibody-based methods. First, HEPACAM's post-translational modifications significantly impact detection; as a glycoprotein, HEPACAM exhibits variable molecular weights ranging from 46-72 kDa depending on glycosylation state . This variability necessitates careful consideration of sample processing methods, with deglycosylation treatments sometimes required for consistent band visualization in Western blot applications.

Antibody clone selection represents another major source of variability. Different clones recognize distinct epitopes; for instance, clone 419305 targets the Val34-Tyr242 region of human HEPACAM , while other antibodies may target cytoplasmic versus extracellular domains, influencing detection patterns. The subcellular localization of HEPACAM also affects detection outcomes, as the protein distributes differently between cell-cell contacts, cytoplasmic regions, and membrane surfaces depending on cellular context and activation state .

Temporal expression patterns introduce additional variability, with HEPACAM expression in developing brain structures changing dramatically over time. In human cerebral cortical spheroids, HEPACAM immunopanning efficiency increases substantially after 100 days of differentiation, reflecting delayed astrocyte generation following neurogenesis . This developmental timeline must be considered when comparing results across timepoints. Tissue preparation methods also impact detection sensitivity; for paraffin-embedded specimens, heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes has proven effective , while different protocols may be optimal for frozen sections or cell suspensions. Awareness of these variables enables researchers to design more consistent, reproducible HEPACAM detection protocols across experimental conditions.

How can researchers address non-specific binding when using FITC-conjugated HEPACAM antibodies?

Non-specific binding represents a significant challenge when working with FITC-conjugated HEPACAM antibodies. Researchers can implement several strategies to minimize this issue and improve signal specificity. First, optimization of blocking conditions is essential; increasing the concentration of blocking proteins such as bovine serum albumin (BSA) or normal serum from the same species as the secondary antibody (when using unconjugated primaries) to 5-10% can significantly reduce non-specific interactions. For Western blot applications specifically, 5% non-fat dry milk in TBST has proven effective as a blocking and diluting buffer .

Titration of antibody concentration is equally critical. While manufacturers typically provide recommended dilutions, researchers should perform systematic titration experiments to identify the optimal concentration that maximizes specific signal while minimizing background. For immunohistochemical applications, dilutions around 1/500 (0.994 μg/ml) have been validated for certain HEPACAM antibody clones , but optimal concentrations will vary based on specific applications and sample types.

Including appropriate controls helps distinguish non-specific from specific binding. Isotype controls matched to the primary antibody's host species and immunoglobulin class (e.g., mouse IgG1 for mouse monoclonal antibodies) should be run in parallel with experimental samples . For cell sorting applications using FITC-conjugated HEPACAM antibodies, unstained and single-stained controls are essential for setting appropriate gates and compensation parameters. When persistent non-specific binding occurs despite these measures, pre-absorption of the antibody with the immunizing peptide (when available) can provide definitive evidence of binding specificity. These methodological refinements collectively improve the signal-to-noise ratio and reliability of FITC-conjugated HEPACAM antibody applications.

What approaches can be used to quantify HEPACAM expression in co-culture systems?

Quantifying HEPACAM expression in co-culture systems requires multifaceted approaches that account for cellular heterogeneity. Flow cytometry using FITC-conjugated HEPACAM antibodies offers a powerful method for quantifying expression levels across different cell populations within co-cultures. This approach allows researchers to gate cells based on HEPACAM positivity and simultaneously assess additional markers to distinguish between cell types . For each population, mean fluorescence intensity (MFI) values provide quantitative measures of HEPACAM expression levels that can be compared across experimental conditions.

Western blot analysis with densitometric quantification offers complementary protein-level measurement. In published studies examining HEPACAM and connexin 43 relationships, researchers normalized the densities of target protein bands to loading controls like GAPDH using ImageJ software, then calculated mean relative densities across multiple independent experiments . This approach revealed significant differences in protein expression levels, with statistical validation through one-way ANOVA with Tukey's multiple comparison tests .

For spatial information within co-cultures, quantitative immunofluorescence microscopy provides valuable insights. Co-localization analysis of HEPACAM with other markers can be performed using specialized software to calculate Pearson's correlation coefficients or Manders' overlap coefficients, providing quantitative measures of protein association. When examining HEPACAM's association with connexin 43, researchers used confocal microscopy with a 60× objective to visualize co-localization (appearing as yellow fluorescence in merged images) particularly at cell-cell contacts . This approach, combined with quantitative image analysis, enables comprehensive assessment of both expression levels and functional interactions in complex co-culture systems.

How can researchers interpret discrepancies between HEPACAM detection methods in developmental studies?

Discrepancies between different HEPACAM detection methods in developmental studies often arise from methodological and biological variables that require careful interpretation. Researchers should consider developmental timing as a primary factor; in human cerebral cortical spheroids, HEPACAM immunopanning efficiency increases dramatically after 100 days of differentiation, reflecting the delayed onset of astrocyte generation following neurogenesis . This temporal pattern means that apparent discrepancies between early and late timepoints may reflect genuine biological progression rather than technical inconsistencies.

Detection method sensitivity thresholds also contribute to apparent discrepancies. Flow cytometry using FITC-conjugated HEPACAM antibodies typically offers greater sensitivity for detecting low-abundance surface expression compared to immunohistochemistry, potentially identifying HEPACAM-positive cells earlier in development. When comparing RNA expression data with protein detection, researchers should acknowledge that transcriptional activation precedes protein accumulation, and post-translational regulation may result in discrepancies between mRNA and protein levels.

Epitope accessibility differences across methodologies represent another potential source of discrepancy. Antibodies targeting different domains of HEPACAM (extracellular versus cytoplasmic) may yield different results depending on protein conformation and molecular interactions. For instance, HEPACAM's association with connexin 43 at cell-cell contacts could potentially mask certain epitopes in intact tissues while remaining accessible in dissociated cell preparations .

To resolve these discrepancies, researchers should implement comprehensive approaches combining multiple detection methods. RNA-seq transcriptional profiling of HEPACAM-isolated cells can verify expression of established astrocyte markers like Aquaporin-4 , while parallel protein-level analyses provide complementary data. Cross-validation across methods, combined with appropriate developmental timeline considerations, enables more accurate interpretation of HEPACAM expression patterns throughout neural development.