ICAM5 Antibody

What is ICAM5 and why is it important in neuroscience research?

ICAM5 (Intercellular Adhesion Molecule 5), also known as Telencephalin, is a type I integral membrane glycoprotein of the immunoglobulin superfamily. It is exclusively expressed in telencephalic neurons, specifically in excitatory neuronal cell bodies, dendritic shafts, and dendritic filopodia . ICAM5 plays critical roles in:

• Dendritic outgrowth and spine maturation

• Organization and stabilization of dendritic spines important for synaptic plasticity

• Neural communication and development of synaptic structures

• Regulation of microglia-neuron interactions

• Neuroprotection during inflammatory responses

ICAM5 functions in both the immune and nervous systems, making it an important target for studying neuroinflammatory conditions and synaptic development .

What are the common applications for ICAM5 antibodies in research?

ICAM5 antibodies are used in multiple experimental applications:

The optimal dilution should be determined experimentally for each application and antibody. For example, the antibody ABIN7258626 is recommended at 1:50 for Western blot applications , while others may perform optimally at higher dilutions such as 1:500-1:3000 .

How should ICAM5 expression patterns be interpreted in brain tissue?

When interpreting ICAM5 staining in brain tissue:

• Expect telencephalon-specific expression (cerebral cortex, hippocampus, striatum)

• ICAM5 is predominantly expressed in postnatal excitatory neurons

• The protein localizes to neuronal cell bodies, dendritic shafts, and dendritic filopodia

• Expression increases during postnatal development

• Expression is absent in ICAM5 knockout mice (useful as negative controls)

In immunohistochemical analysis, ICAM5 typically shows punctate staining along dendrites with stronger labeling at dendritic spines. Verification using ICAM5 knockout tissue is crucial for confirming antibody specificity .

What is the expected molecular weight of ICAM5 and why does it sometimes appear differently on Western blots?

While the calculated molecular weight of ICAM5 is approximately 97 kDa, the observed molecular weight on Western blots typically ranges from 115-140 kDa, with some reports showing bands at 146-180 kDa . This discrepancy is due to:

• Post-translational modifications: Extensive glycosylation increases apparent molecular weight

• Multiple isoforms: Different splice variants may be detected

• Proteolytic processing: ICAM5 can be cleaved by matrix metalloproteases

Specific examples from the research literature show:

• Human brain (motor cortex) tissue shows specific ICAM5 bands at approximately 115 and 140 kDa under reducing conditions

• Simple Western analysis detected ICAM5 at approximately 146 and 180 kDa in human brain tissue

• The observed molecular weight is consistently reported as 140 kDa in validated antibodies

When validating a new ICAM5 antibody, researchers should expect multiple bands within this range rather than a single band at the calculated 97 kDa size.

How can I validate the specificity of an ICAM5 antibody?

A comprehensive ICAM5 antibody validation protocol should include:

• Positive and negative tissue controls:

  • Positive: Telencephalic regions (cerebral cortex, hippocampus)

  • Negative: Non-telencephalic regions (cerebellum, brain stem)

• Genetic controls:

  • Compare staining between wild-type and ICAM5 knockout animals

  • The ICAM-5cp antibody has been validated using this approach

• Epitope blocking:

  • Pre-incubate antibody with the immunizing peptide

  • Observe reduction or elimination of signal

• Multiple detection methods:

  • Compare results across WB, IHC, and ICC

  • Confirm consistent patterns of expression

• Cross-validation with multiple antibodies:

  • Use antibodies targeting different epitopes (N-terminal vs. C-terminal)

  • Compare commercial antibodies from different vendors

As demonstrated in research by Yoshihara et al. (1994), the ICAM-5cp antibody specifically recognized ICAM5 in wild-type but not ICAM5 knockout animals, confirming its specificity .

What are the optimal sample preparation methods for detecting ICAM5 in brain tissue?

For optimal ICAM5 detection in brain tissue:

For Western Blotting:

• Rapidly extract brain tissue and freeze in liquid nitrogen

• Homogenize in RIPA or NP-40 buffer containing protease inhibitors

• Use reducing conditions with β-mercaptoethanol or DTT

• Load 20-50 μg of total protein per lane

• Transfer to PVDF membrane (preferred over nitrocellulose for high molecular weight proteins)

For Immunohistochemistry:

• Perfusion fixation with 4% paraformaldehyde is recommended

• For paraffin embedding, heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes significantly improves signal

• For frozen sections, post-fixation in cold acetone may preserve antigenicity

• Block with appropriate serum (e.g., 5% normal goat serum) to reduce background

For Immunoprecipitation:

• Use mild lysis conditions to preserve protein-protein interactions

• Pre-clear lysates with protein A/G beads

• Incubate with ICAM5 antibody (5-10 μg per mg of protein)

• Capture complexes with protein A/G beads

• Wash stringently to reduce non-specific binding

These methods have been validated in studies examining ICAM5's interactions with integrin β1 and β2 in brain homogenates .

How can ICAM5 antibodies be used to study neuron-microglia interactions?

ICAM5 plays a significant role in regulating neuron-microglia interactions, particularly through its soluble form. Research methodologies include:

• Co-culture systems:

  • Primary neurons with microglia

  • Analyze effects of ICAM5 antibody blocking on interactions

  • The cleaved soluble form of ICAM5 has been shown to bind to microglia and co-localize with β2 integrin

• ICAM5 immunoprecipitation from brain tissue:

  • Can detect significant amounts of β2 integrin in the immune-complex

  • BV-2 microglial cells treated with neuron-conditioned medium show association of ICAM5 with both β1 and β2 integrins

• Functional assays:

  • Microglia adhesion assays on ICAM5-coated surfaces

  • Phagocytosis assays in the presence of soluble ICAM5

  • Cytokine secretion measurements (ELISA for TNF-α, IL-1β, IL-10)

Key research findings show that soluble ICAM5 reduces microglia adhesion and phagocytosis while altering cytokine production. Specifically, soluble ICAM5 D1-9 significantly reduces TNF-α secretion (p<0.05) and increases anti-inflammatory IL-10 production in LPS-stimulated microglia .

What approaches are effective for studying soluble ICAM5 (sICAM5) in biological fluids?

Studying sICAM5 in biological fluids requires specialized techniques:

• Sample preparation:

  • For cerebrospinal fluid (CSF): Collect with protease inhibitors and process immediately

  • For cell culture supernatants: Concentrate using centrifugal filters

  • For serum/plasma: Remove cells and platelets by centrifugation

• Detection methods:

  • Sandwich ELISA using capture/detection antibody pairs

    • Example: Mouse Anti-Human ICAM-5 Monoclonal Antibody (MAB1950) as capture antibody with Goat Anti-Human ICAM-5 Biotinylated Antigen Affinity-purified Polyclonal Antibody (BAF1950) for detection

  • Western blotting of concentrated samples

  • Immunoprecipitation followed by mass spectrometry

• Quantification strategies:

  • Standard curves with recombinant ICAM5 proteins

  • Relative quantification comparing disease vs. control samples

  • Normalization to total protein content

Research has shown that cerebrospinal fluid from patients with progressive forms of multiple sclerosis contains decreased levels of sICAM5, suggesting impairment of this endogenous protective pathway .

How can ICAM5 antibodies be used to investigate ICAM5 cleavage by matrix metalloproteases?

ICAM5 cleavage by matrix metalloproteases (MMPs) is a crucial regulatory mechanism. Effective research approaches include:

• In vitro cleavage assays:

  • Recombinant ICAM5 incubated with purified MMPs

  • Analysis of cleavage products by Western blotting using domain-specific antibodies

  • Identification of cleavage sites by mass spectrometry

• Cell-based assays:

  • Neuronal cultures treated with NMDA to induce ICAM5 cleavage

  • Collection of conditioned medium for soluble ICAM5 detection

  • Western blotting of cellular fractions to track membrane-bound and shed forms

• Inhibitor studies:

  • Treatment with MMP inhibitors to block cleavage

  • Analysis of functional consequences on dendritic spine maturation

  • Comparison between wild-type and MMP-9 knockout animals

Research has demonstrated that MMP-9 plays a role in the developmental processing of ICAM5, as evidenced by changes in ICAM5 expression in MMP-9 null animals . Additionally, neurons stimulated with LPS or splenocyte supernatant upregulate both ICAM5 and MMP-9, mimicking inflammatory conditions .

What are the methodological considerations for studying ICAM5 in neurodevelopmental disorders?

When investigating ICAM5 in neurodevelopmental disorders:

• Animal models:

  • ICAM5 knockout mice show altered dendritic spine development

  • Fmr1 knockout mice (Fragile X syndrome model) can be used to study ICAM5 mRNA binding by FMRP

  • RNA-binding protein immunoprecipitation (RIP) can identify FMRP binding sites on ICAM5 mRNA

• Primary neuron cultures:

  • From wild-type vs. disease model animals

  • Comparison of ICAM5 expression, localization, and processing

  • Examination of dendritic spine morphology and density

• Human sample analysis:

  • Post-mortem brain tissue from patients vs. controls

  • CSF analysis for soluble ICAM5 levels

  • Genetic association studies for ICAM5 variants

• Molecular techniques:

  • Crosslinking-Immunoprecipitation and High-Throughput sequencing (HITS-CLIP)

  • MEME and Dreme software for motif analysis

  • Tomtom software to compare existing motifs in databases

Research in Fragile X syndrome has utilized RNA-binding protein immunoprecipitation to investigate FMRP binding to ICAM5 mRNA, providing insights into post-transcriptional regulation of ICAM5 in this neurodevelopmental disorder .

Why might I observe inconsistent ICAM5 antibody staining in brain tissue sections?

Inconsistent ICAM5 staining can result from several factors:

• Fixation issues:

  • Over-fixation can mask epitopes

  • Under-fixation can cause protein degradation

  • Solution: Optimize fixation time (typically 24-48 hours for adult brain)

• Antigen retrieval problems:

  • Insufficient retrieval for paraffin sections

  • Solution: Use Tris-EDTA buffer (pH 9.0) for 20 minutes as demonstrated in successful ICAM5 IHC protocols

• Antibody specificity:

  • Some antibodies target specific domains that may be inaccessible

  • Solution: Compare N-terminal vs. C-terminal targeting antibodies

• Regional variation:

  • ICAM5 is telencephalon-specific

  • Solution: Include positive control regions (cerebral cortex) alongside experimental regions

• Developmental stage differences:

  • ICAM5 expression increases postnatally

  • Solution: Age-match samples carefully

For optimal results, researchers should include both positive controls (cortical tissue) and negative controls (cerebellum or ICAM5 knockout tissue) in each experiment, as demonstrated in successful immunohistochemical studies .

How can I address non-specific bands in Western blots using ICAM5 antibodies?

When encountering non-specific bands:

• Antibody validation:

  • Test the antibody on ICAM5 knockout tissue/cells as negative control

  • Use competing peptides to confirm specificity

  • Compare results with multiple antibodies targeting different epitopes

• Sample preparation optimization:

  • Include additional protease inhibitors to prevent degradation

  • Adjust lysis buffer composition (RIPA vs. milder NP-40 buffers)

  • Optimize protein loading (20-50 μg typically optimal)

• Technical adjustments:

  • Increase washing stringency (higher salt concentration, longer washes)

  • Optimize blocking conditions (5% non-fat milk vs. BSA)

  • Adjust antibody concentration (serial dilutions between 1:500-1:3000)

  • Try different membrane types (PVDF often superior for high MW proteins)

• Data interpretation:

  • Expect multiple specific bands (115-140 kDa and 146-180 kDa range)

  • Different bands may represent glycosylation variants or proteolytic products

  • Compare with published literature showing multiple specific bands

Remember that ICAM5 typically appears at a higher molecular weight than calculated (observed ~140 kDa vs. calculated 97 kDa) due to post-translational modifications .

What strategies can address poor signal-to-noise ratio in ICAM5 immunofluorescence?

To improve signal-to-noise ratio in ICAM5 immunofluorescence:

• Fixation optimization:

  • Compare 4% PFA vs. methanol fixation

  • Optimize fixation duration (over-fixation masks epitopes)

• Blocking enhancement:

  • Extend blocking time (2 hours to overnight)

  • Try different blocking agents (5% normal serum, 3% BSA, commercial blockers)

  • Include detergents (0.1-0.3% Triton X-100) for improved penetration

• Antibody incubation:

  • Incubate primary antibody at 4°C overnight

  • Optimize antibody concentration through titration

  • Consider using signal amplification systems (tyramide signal amplification)

• Background reduction:

  • Add 0.1-0.3% Tween-20 to wash buffers

  • Increase number and duration of washes

  • Pre-adsorb secondary antibodies with tissue powder

• Advanced imaging techniques:

  • Use confocal microscopy for improved signal resolution

  • Apply deconvolution algorithms to reduce out-of-focus signal

  • Consider spectral unmixing for overlapping fluorophores

These strategies have proven effective in visualizing ICAM5 and its colocalization with integrins in primary microglia cultures .

How can ICAM5 antibodies contribute to multiple sclerosis and neuroinflammation research?

ICAM5 antibodies offer valuable tools for multiple sclerosis (MS) research:

• Neuroprotective mechanisms:

  • ICAM5 knockout mice show more severe EAE (experimental autoimmune encephalomyelitis) in the chronic phase, indicating ICAM5's neuroprotective function

  • Soluble ICAM5 application ameliorates EAE symptoms, suggesting therapeutic potential

  • Cerebrospinal fluid from progressive MS patients shows decreased sICAM5 levels

• Methodological approaches:

  • Intrathecal application of recombinant sICAM5 in EAE models

  • Quantification of sICAM5 in patient CSF samples

  • Analysis of T cell-neuron contacts mediated by ICAM5-LFA-1 interactions

• Experimental models:

  • MOG35-55 peptide-induced EAE in wild-type vs. ICAM5-/- mice

  • In vitro neuroinflammatory conditions using LPS or cytokine stimulation

  • Primary neuron-microglia co-culture systems

Research has shown that soluble ICAM5 can reduce pro-inflammatory cytokine production (TNF-α, IL-1β) while increasing anti-inflammatory IL-10 in LPS-stimulated microglia, suggesting ICAM5 might suppress the M1 microglial response or tilt the phenotype toward M2 .

What are the latest methodological advances in studying ICAM5 protein-protein interactions?

Cutting-edge techniques for studying ICAM5 interactions include:

• Proximity-based labeling methods:

  • BioID or TurboID fusion proteins to identify proximity partners

  • APEX2 for electron microscopy visualization of interaction sites

  • Split-BioID for detecting specific protein-protein interactions

• Advanced microscopy techniques:

  • Super-resolution microscopy (STORM, PALM) for nanoscale localization

  • Förster resonance energy transfer (FRET) to confirm direct interactions

  • Single-molecule tracking to study dynamics of ICAM5 interactions

• Functional protein arrays:

  • Recombinant ICAM5 domains on protein arrays

  • Screening for novel binding partners

  • Characterization of binding affinities and specificities

• Computational approaches:

  • Molecular docking to predict interaction interfaces

  • Molecular dynamics simulations to study interaction dynamics

  • Machine learning for predicting interaction networks

Research has successfully used recombinant ICAM5-Fc domains coupled to beads to study microglia binding, revealing sequential recruitment of β2 and β1 integrins during different stages of interaction .

How can ICAM5 antibodies be utilized in investigating synaptic plasticity mechanisms?

ICAM5 antibodies provide powerful tools for studying synaptic plasticity:

• Developmental studies:

  • Tracking ICAM5 expression during critical periods of synaptogenesis

  • Correlating ICAM5 levels with dendritic spine maturation

  • Comparing wild-type vs. knockout animals for spine density and morphology

• Activity-dependent regulation:

  • NMDA receptor activation leads to ICAM5 shedding from neuronal membranes

  • Antibodies against different ICAM5 domains can track intact vs. cleaved forms

  • Time-course analysis following synaptic activation

• Functional manipulation:

  • Function-blocking antibodies to interfere with ICAM5-mediated cell adhesion

  • Domain-specific antibodies to block particular interactions

  • Antibody-induced clustering to mimic ligand binding

• Synaptic localization:

  • Super-resolution imaging of ICAM5 distribution at synapses

  • Co-localization with synaptic markers

  • Immunoelectron microscopy for ultrastructural localization

Research has demonstrated that ICAM5 is solubilized from NMDA-treated neurons and affects dendritic filopodia formation, suggesting a role in activity-dependent synaptic remodeling . Function-blocking antibodies against ICAM5 have successfully been used to demonstrate its role in neurite outgrowth .