FABP7 Antibody

What is FABP7 and why is it important in neuroscience research?

FABP7 is a member of the fatty acid binding protein family that regulates intracellular lipid metabolism. It binds to long-chain fatty acids and plays important roles in transporting fatty acids into cells. In the central nervous system, FABP7 is highly expressed in brain tumors, particularly gliomas, and has been implicated in various neural processes .

FABP7 has gained significant attention because it is overexpressed in many types of tumors including brain, breast, colorectal, and prostate cancers. Research indicates it plays crucial roles in tumor development, with particularly strong evidence for its involvement in glioblastoma cell proliferation and migration .

How specific are commercially available FABP7 antibodies?

Commercial FABP7 antibodies demonstrate high specificity with minimal cross-reactivity to other FABP family members. For example, some polyclonal antibodies show less than 5% cross-reactivity with related proteins including FABP1, FABP2, FABP3, FABP4, FABP5, FABP6, FABP8, and FABP9 .

Validation studies typically include Western blot analysis using both recombinant proteins and tissue lysates from cerebellum and hippocampus, where FABP7 is naturally expressed. Additionally, the specificity of FABP7 antibodies has been confirmed using tissues from Fabp7 knockout mice compared to wild-type controls, which provides definitive evidence of antibody specificity .

What are the recommended applications for FABP7 antibodies?

FABP7 antibodies have been successfully validated for multiple applications, including:

• Western blotting (detecting bands at approximately 18 kDa)

• Immunohistochemistry (both paraffin-embedded and frozen sections)

• Immunocytochemistry

• Simple Western assays

• Immunoprecipitation

• ELISA

These antibodies work particularly well in neural tissues such as cerebellum and hippocampus, as well as in tumor samples, especially gliomas and melanomas . When selecting an antibody, researchers should consider their specific application and target tissue, as some antibodies may perform better in certain contexts than others.

How should I design experiments to evaluate FABP7 expression in glioma samples?

When designing experiments to evaluate FABP7 expression in glioma samples, a multi-modal approach is recommended:

• Tissue preparation: For immunohistochemistry, use 4% paraformaldehyde fixation followed by paraffin embedding or cryoprotection depending on your specific protocol .

• Antibody selection: Choose antibodies validated specifically for glioma tissues. Polyclonal antibodies like those used in published studies (e.g., rabbit anti-Fabp7 at 1:1000 dilution) have shown reliable results .

• Controls: Include both positive controls (known FABP7-expressing tissues like cerebellum) and negative controls (Fabp7 knockout tissue if available, or primary antibody omission) .

• Expression analysis: Complement protein detection with mRNA analysis using qPCR. Established studies have used reference genes such as Ywhaz, Ppia, Ywhah, or Sdha for normalization .

• Clinical correlation: To maximize research value, correlate FABP7 expression with clinical parameters such as tumor grade, patient survival, and treatment response .

What protocols are most effective for siRNA knockdown of FABP7 in oligodendrocyte precursor cells (OPCs)?

For effective siRNA knockdown of FABP7 in OPCs, the following protocol has been validated in research settings:

• Cell preparation: Culture isolated OPCs from mixed glial cell cultures for 2 days in vitro (DIV) with appropriate growth factors.

• Pre-transfection: Change to OPC medium without antibiotics overnight to improve transfection efficiency.

• Transfection: Use 50 nM FABP7 siRNA with 1% Lipofectamine siRNAMAX diluted in Opti-MEM according to manufacturer protocols. Always include a non-targeting control siRNA.

• Post-transfection: After 6 hours, replace the medium with OPC medium without growth factors, thyroxine, and triiodothyronine to promote differentiation.

• Analysis timeline: Evaluate knockdown efficiency and phenotypic effects 48 hours after transfection .

• Validation methods: Confirm knockdown by Western blot, qPCR, and immunocytochemistry to ensure both protein and mRNA levels are reduced.

This protocol has been shown to effectively reduce FABP7 expression and allows for the assessment of its functional role in OPC differentiation.

How can I effectively use FABP7 antibodies in immunohistochemistry of brain sections?

For optimal immunohistochemical staining of FABP7 in brain sections:

• Tissue preparation:

  • For paraffin-embedded sections: Fix tissues in 4% paraformaldehyde, process through graded alcohols, embed in paraffin, and cut 5-10 μm sections.

  • For frozen sections: After fixation, cryoprotect in graded sucrose solutions (15-30%), embed in OCT compound, and cut 30 μm sections .

• Antigen retrieval: For paraffin sections, heat-mediated antigen retrieval in citrate buffer (pH 6.0) is typically effective.

• Blocking and permeabilization:

  • Block endogenous peroxidase activity with 3% H₂O₂ in TBS for 30 minutes.

  • Wash with TBST (0.05% Tween-20 in TBS) 3 times for 10 minutes each.

  • Block with 2-5% normal serum (matching the species of the secondary antibody) for 1 hour .

• Antibody incubation:

  • Primary antibody: Incubate with rabbit anti-FABP7 antibody (1:1000 dilution) overnight at 4°C.

  • Secondary antibody: After washing, incubate with HRP-conjugated secondary antibody (1:100) at room temperature .

• Detection and visualization:

  • For chromogenic detection, use DAB substrate kits.

  • For fluorescent detection, use appropriate fluorophore-conjugated secondary antibodies.

  • Counterstain nuclei with hematoxylin (for chromogenic) or DAPI (for fluorescent) .

• Controls: Include technical controls (primary antibody omission) and biological controls (FABP7 knockout tissue if available) .

How should I interpret FABP7 expression patterns in relation to glioma progression and prognosis?

When interpreting FABP7 expression in glioma samples, consider these evidence-based guidelines:

What are the potential confounding factors when assessing FABP7 antibody staining in research samples?

When evaluating FABP7 antibody staining, researchers should account for these potential confounding factors:

To address these confounders, always include appropriate controls, standardize protocols, and use multiple detection methods when possible.

How can I resolve conflicting FABP7 expression data between mRNA and protein levels?

Discrepancies between FABP7 mRNA and protein levels are not uncommon. To resolve such conflicts:

• Technical verification:

  • Confirm antibody specificity using multiple antibodies targeting different epitopes

  • Validate qPCR primers using standard curves and melt curve analysis

  • Include appropriate reference genes for normalization (e.g., Ywhaz, Ppia, Ywhah, or Sdha)

• Post-transcriptional regulation: Consider mechanisms that may explain discrepancies:

  • microRNA-mediated suppression of translation

  • Alterations in protein stability and degradation

  • Subcellular localization changes affecting protein extraction efficiency

• Temporal dynamics: mRNA and protein may have different half-lives. Conduct time-course experiments to capture temporal relationships between transcript and protein levels.

• Cell-type heterogeneity: In tissue samples, different cell populations may contribute differently to bulk measurements. Consider single-cell approaches or cell-type enrichment strategies.

• Validation approaches:

  • Use siRNA knockdown to confirm antibody specificity by demonstrating reduced signal

  • Compare findings with public database expression data from CGGA, TCGA, and GEO datasets

  • Employ complementary techniques like in situ hybridization to localize mRNA

What is the relationship between FABP7 and angiogenesis in glioma, and how can antibody-based techniques help investigate this?

FABP7 has been implicated in angiogenesis pathways in glioma through several lines of evidence:

• Pathway correlation: Gene Set Enrichment Analysis (GSEA) has demonstrated that FABP7 expression is significantly associated with angiogenesis pathways. FABP7 shows strong correlation coefficients (>0.4) with seven key angiogenic factors: POSTN, TIMP1, PDGFA, FGFR1, S100A4, COL5A2, and STC1 .

• Treatment response: Immunohistochemical studies revealed that higher FABP7 expression correlates with poorer response to antiangiogenic therapy (apatinib), suggesting FABP7 may confer resistance to anti-angiogenic treatment .

Antibody-based techniques to investigate this relationship include:

• Multiplex immunofluorescence: Co-localize FABP7 with angiogenic markers in the same tissue sections to establish spatial relationships

• Proximity ligation assay: Detect protein-protein interactions between FABP7 and angiogenic factors

• Chromatin immunoprecipitation (ChIP): Determine if FABP7 regulates the expression of angiogenic genes

• Reverse Phase Protein Arrays (RPPA): Quantitatively assess FABP7 and multiple angiogenic factors simultaneously in large sample sets

How can FABP7 antibodies be used to study its role in neurodevelopmental processes such as myelination?

FABP7 antibodies are valuable tools for investigating its role in neurodevelopmental processes, particularly myelination:

• Developmental expression profiling: Immunohistochemistry with FABP7 antibodies can track expression patterns throughout postnatal development. Research has shown that FABP7 expression closely follows the timeline of myelination during postnatal development .

• Cell-type specific localization: Double immunostaining with FABP7 antibodies and cell-type specific markers can identify which neural cell populations express FABP7 during different developmental stages.

• Functional studies:

  • siRNA knockdown of FABP7 in oligodendrocyte precursor cells (OPCs) has demonstrated its requirement for proper OPC differentiation in vitro

  • Comparing wild-type and Fabp7 knockout animals showed a transient delay in developmental myelination, with full myelination being established before adulthood

• Remyelination studies: Although FABP7 is important for initial myelination, antibody-based studies in focal demyelination models revealed that FABP7 is dispensable for remyelination, as knockout of Fabp7 did not alter remyelination efficiency .

• Mechanistic investigations: Immunoprecipitation with FABP7 antibodies can identify binding partners involved in myelination pathways.

What techniques can be used to evaluate FABP7 antibody cross-reactivity with other FABP family members?

To thoroughly evaluate FABP7 antibody cross-reactivity with other FABP family members:

• Direct ELISA screening:

  • Coat plates with recombinant proteins of all FABP family members (FABP1-9)

  • Test antibody binding to each family member

  • Calculate percent cross-reactivity relative to FABP7 binding

  • Published antibodies show less than 5% cross-reactivity with other family members

• Western blot analysis:

  • Run recombinant FABP proteins on SDS-PAGE

  • Probe with the FABP7 antibody

  • Evaluate band presence/absence and intensity for each family member

  • Include tissue samples known to express different FABP members as biological controls

• Knockout validation:

  • Test antibody on tissues from Fabp7 knockout mice

  • Complete absence of signal confirms specificity

  • Residual signal may indicate cross-reactivity

• Peptide competition assays:

  • Pre-incubate antibody with excess specific peptide from FABP7

  • Pre-incubate separate aliquots with peptides from other FABP family members

  • Compare signal reduction to assess specific versus non-specific binding

• Mass spectrometry verification:

  • Perform immunoprecipitation with the FABP7 antibody

  • Analyze precipitated proteins by mass spectrometry

  • Identify all captured proteins to detect any off-target binding

What are common causes of weak or absent FABP7 signal in immunohistochemistry, and how can they be addressed?

When confronted with weak or absent FABP7 signal in immunohistochemistry, consider these potential causes and solutions:

• Fixation issues:

  • Problem: Overfixation can mask epitopes

  • Solution: Optimize fixation time (typically 24 hours for 4% PFA) or use antigen retrieval

• Antibody concentration:

  • Problem: Insufficient primary antibody concentration

  • Solution: Perform titration experiments (try 1:500 to 1:2000 dilutions) to determine optimal concentration

• Antigen retrieval inadequacy:

  • Problem: Insufficient epitope unmasking

  • Solution: Try different antigen retrieval methods (heat-induced in citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Tissue-specific expression levels:

  • Problem: FABP7 expression varies by tissue type and developmental stage

  • Solution: Include positive control tissues known to express high FABP7 levels (e.g., cerebellum)

• Antibody storage/handling:

  • Problem: Antibody degradation

  • Solution: Adhere to storage recommendations; avoid repeated freeze-thaw cycles

• Detection system sensitivity:

  • Problem: Insensitive detection method

  • Solution: Switch to more sensitive detection systems (e.g., polymer-based HRP systems, tyramide signal amplification)

• Endogenous peroxidase activity:

  • Problem: Incomplete blocking of endogenous activity

  • Solution: Extend H₂O₂ blocking step to 30 minutes using 3% H₂O₂ in TBS

How can I optimize FABP7 antibody performance for Western blot applications?

For optimal FABP7 antibody performance in Western blot applications:

• Sample preparation:

  • Use RIPA buffer containing protease and phosphatase inhibitors

  • Homogenize tissues on ice and centrifuge to collect the supernatant

  • Determine protein concentration using BCA Protein Assay

• Gel electrophoresis conditions:

  • Load adequate protein (20 μg total protein is typically sufficient)

  • Use gradient gels (4-20%) for optimal resolution of the approximately 18 kDa FABP7 protein

• Transfer parameters:

  • Use PVDF membranes for optimal protein binding

  • Consider semi-dry transfer systems for small proteins like FABP7

  • Verify transfer efficiency with reversible protein stains

• Blocking conditions:

  • Block with 5% nonfat dry milk in 0.1 M TBST for 1 hour at room temperature

  • Some antibodies may perform better with BSA-based blocking buffers

• Antibody incubation:

  • Primary antibody: Incubate with anti-FABP7 antibody (1:1000 dilution) in 5% nonfat dry milk overnight at 4°C

  • Secondary antibody: Use HRP-conjugated secondary antibody at 1:50-1:100 dilution

• Detection optimization:

  • Use enhanced chemiluminescence (ECL) with exposure time optimization

  • For weak signals, consider higher sensitivity ECL substrates

• Controls:

  • Include recombinant FABP7 protein as a positive control

  • Use tissue lysates known to express FABP7 (cerebellum, hippocampus)

  • Include lysates from Fabp7 knockout tissues as negative controls when available

What strategies can resolve non-specific binding when using FABP7 antibodies in immunoprecipitation?

To minimize non-specific binding in FABP7 immunoprecipitation experiments:

• Pre-clearing the lysate:

  • Incubate lysates with Protein A/G beads without antibody for 1 hour

  • Remove beads by centrifugation before adding the FABP7 antibody

  • This reduces proteins that bind non-specifically to the beads

• Antibody selection:

  • Use antibodies specifically validated for immunoprecipitation

  • Consider using monoclonal antibodies for higher specificity

  • If using polyclonal antibodies, purify by antigen-affinity chromatography

• Buffer optimization:

  • Adjust salt concentration in wash buffers (150-500 mM NaCl)

  • Add mild detergents (0.1-0.5% NP-40 or Triton X-100)

  • Include carrier proteins (BSA) to reduce non-specific interactions

• Cross-linking strategies:

  • Cross-link antibodies to beads using dimethyl pimelimidate (DMP)

  • This prevents antibody co-elution and reduces background in downstream applications

• Elution conditions:

  • Use specific peptide elution instead of harsh denaturing conditions

  • Gradient elution with increasing stringency can separate specific from non-specific interactions

• Negative controls:

  • Include isotype control antibodies matched to your FABP7 antibody

  • Use lysates from Fabp7 knockout tissues when available

• Validation of results:

  • Confirm pulled-down proteins by Western blot and/or mass spectrometry

  • Compare band patterns between specific and control immunoprecipitations

How might FABP7 antibodies be utilized in developing novel diagnostic approaches for glioma?

FABP7 antibodies show significant potential for novel glioma diagnostic approaches:

• Liquid biopsy development:

  • FABP7 antibodies could be used to detect circulating FABP7 protein in serum or cerebrospinal fluid

  • This could provide minimally invasive biomarkers for glioma diagnosis and monitoring

• Immunohistochemical classification systems:

  • FABP7 expression levels could be incorporated into multi-marker panels

  • Research shows FABP7 expression correlates with clinicopathological features and could refine current glioma classification systems

• Theranostic applications:

  • Radiolabeled FABP7 antibodies could enable simultaneous imaging and therapeutic targeting

  • This approach could identify patients likely to benefit from FABP7-targeted therapies

• Early detection strategies:

  • Given the correlation between FABP7 and patient prognosis, antibody-based early detection methods might identify aggressive tumors before clinical symptoms appear

• Predictive biomarker development:

  • FABP7 immunostaining could predict response to antiangiogenic therapies like apatinib

  • Research demonstrates that higher FABP7 expression correlates with poorer response to such treatments

• Multiplexed diagnostic approaches:

  • Combining FABP7 antibodies with antibodies against the seven correlated angiogenic factors (POSTN, TIMP1, PDGFA, FGFR1, S100A4, COL5A2, and STC1) could provide more comprehensive diagnostic profiles

What emerging technologies might enhance the utility of FABP7 antibodies in research and clinical applications?

Several emerging technologies show promise for enhancing FABP7 antibody applications:

• Single-cell antibody-based technologies:

  • Single-cell Western blotting could profile FABP7 expression in individual cells within heterogeneous tumor samples

  • Mass cytometry (CyTOF) with FABP7 antibodies could simultaneously analyze dozens of parameters at single-cell resolution

• Spatial transcriptomics integration:

  • Combining FABP7 immunohistochemistry with spatial transcriptomics could map relationships between FABP7 protein expression and global gene expression patterns in situ

• Extracellular vesicle (EV) analysis:

  • FABP7 antibodies could detect FABP7 in tumor-derived EVs, potentially offering minimally invasive diagnostic approaches

• Nanobody development:

  • Developing smaller antibody fragments (nanobodies) against FABP7 could improve tissue penetration and reduce immunogenicity for in vivo applications

• CRISPR-based diagnostics:

  • CRISPR-Cas systems coupled with FABP7 antibodies could create ultra-sensitive detection platforms

• Live-cell imaging:

  • Cell-permeable FABP7 antibody derivatives could enable real-time tracking of FABP7 dynamics in living cells

• Antibody-drug conjugates (ADCs):

  • Given FABP7's overexpression in glioma, ADCs targeting FABP7 could deliver therapeutic payloads specifically to tumor cells

• Microfluidic applications:

  • Microfluidic antibody arrays could enable rapid, high-throughput FABP7 detection with minimal sample volumes

These emerging technologies could significantly expand the utility of FABP7 antibodies beyond current applications.

How do different FABP7 antibody detection methods compare in sensitivity and specificity?

Key considerations when selecting a detection method:

• Western blot offers excellent specificity for FABP7, consistently detecting the expected 18 kDa band in neural tissues and showing complete absence in Fabp7 knockout samples .

• Immunohistochemistry provides critical spatial information and has been successfully used to correlate FABP7 expression with clinical outcomes in glioma patients .

• Simple Western automated systems show excellent consistency and have been validated for FABP7 detection in human cerebellum and hippocampus tissues .

• Each method has complementary strengths, and combining multiple approaches provides the most comprehensive analysis of FABP7 expression and function.

What are the key differences between mouse and human FABP7 that researchers should consider when designing experiments?

When designing experiments involving FABP7 across species, researchers should consider these key differences between mouse and human FABP7:

• Sequence homology:

  • Mouse and human FABP7 share approximately 92% amino acid identity

  • Most antibodies recognize both species, but epitope-specific antibodies may show species preferences

• Expression patterns:

  • While both species express FABP7 in neural tissues, the temporal and spatial patterns differ

  • In mice, FABP7 expression closely follows the timeline of myelination during postnatal development

  • Human expression patterns may show different developmental timing

• Regulatory mechanisms:

  • Transcriptional regulation of FABP7 may differ between species

  • Response elements in promoter regions show some species-specific variations

• Experimental models:

  • Fabp7 knockout mice show only transient developmental myelination delay with full recovery by adulthood

  • This suggests potentially different compensatory mechanisms between species

• Pathological relevance:

  • In humans, FABP7 overexpression strongly correlates with poor prognosis in glioma

  • Mouse models may not fully recapitulate all aspects of human FABP7-associated pathology

• Antibody validation requirements:

  • Cross-reactivity testing should include both species when possible

  • Controls (especially knockout tissues) from the appropriate species should be used

These considerations are crucial for the appropriate design and interpretation of cross-species FABP7 studies.