FABP7 Antibody, HRP conjugated

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

FABP7 (Fatty Acid Binding Protein 7), also known as B-FABP or BLBP, belongs to a family of small, highly conserved cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. It plays crucial roles in fatty acid uptake, transport, and metabolism in the central nervous system. FABP7 is abundantly expressed in neural stem cells and astrocytes of developing brain, suggesting its importance in neurogenesis and glial cell development . Research has established FABP7 as a downstream gene of the Pax6 transcription factor, essential for maintaining neuroepithelial cells during early cortical development . In pathological conditions such as spinal cord injury, FABP7 expression becomes significantly upregulated in reactive astrocytes, indicating its potential role in neural repair mechanisms .

What are the typical applications for FABP7 antibodies in neuroscience research?

FABP7 antibodies have multiple validated applications in neuroscience research, including Western blotting, immunohistochemistry (both paraffin-embedded and frozen sections), Simple Western assays, and immunocytochemistry . These antibodies enable researchers to detect FABP7 expression in various neural tissues, particularly in studies investigating glial cell biology, neural development, and response to injury. The detection of FABP7 helps in identifying specific cell populations such as neural stem cells, radial glia, oligodendrocyte progenitor cells, and astrocytes. HRP-conjugated versions provide direct detection capability, eliminating the need for secondary antibodies in Western blotting and immunohistochemistry applications, which can enhance sensitivity and reduce background in certain experimental contexts .

What cell types express FABP7 and how can they be identified using immunohistochemistry?

In the normal cortex, approximately 21.6% of FABP7-positive cells are GFAP-positive astrocytes, while about 62% are NG2-positive oligodendrocyte progenitor cells (OPCs) . FABP7 expression has been confirmed in OPCs through co-staining with PDGFRα, another specific marker for these cells . Notably, around 97.1% of NG2-positive OPCs and 82.4% of GFAP-positive astrocytes in the intact cortex express FABP7 . For immunohistochemical detection, tissue sections can be stained with FABP7 antibodies (typically at 5 μg/mL concentration) followed by appropriate visualization systems such as HRP-DAB for brightfield microscopy . Double immunofluorescence labeling with cell-type-specific markers allows precise identification of FABP7-expressing cell populations, revealing its differential expression across neural cell types .

How should researchers optimize immunohistochemistry protocols for FABP7 detection in various tissue preparations?

Optimal immunohistochemistry protocols for FABP7 detection require careful consideration of tissue preparation, antigen retrieval, and antibody incubation conditions. For paraffin-embedded sections, such as human melanoma tissues used in validation studies, immersion fixation followed by deparaffinization and rehydration is recommended . Antigen retrieval methods vary by tissue type, but heat-induced epitope retrieval using citrate buffer (pH 6.0) is commonly effective. When using HRP-conjugated FABP7 antibodies, researchers should incorporate a peroxidase quenching step (typically 3% hydrogen peroxide) to minimize background staining. For optimal results in neural tissues, an antibody concentration of 5 μg/mL with overnight incubation at 4°C has been validated, followed by visualization using DAB chromogen and hematoxylin counterstaining . For double immunofluorescence labeling, sequential staining protocols may be necessary to avoid cross-reactivity, particularly when co-staining for FABP7 and GFAP in reactive astrocytes .

What approaches can researchers use to quantify FABP7 expression changes in injury models?

Quantification of FABP7 expression changes in injury models requires multi-faceted approaches. Western blot analysis represents a primary method, where FABP7 protein levels can be detected at approximately 15-18 kDa and quantified through densitometry . In spinal cord injury models, FABP7 shows significant upregulation after injury, reaching maximum expression at 14 days post-injury . For tissue-level analysis, immunofluorescence staining followed by semi-quantitative measurement of immunoreactivity provides valuable spatial information. This approach has demonstrated increased FABP7 expression in reactive astrocytes after injury, correlating with enhanced GFAP expression . For cellular-level quantification, researchers should employ stereological counting methods to determine the proportion of different cell types expressing FABP7 before and after injury. This approach has revealed significant changes in FABP7+/GFAP+ cell populations in injury models, providing insights into the cell-type-specific response to CNS trauma .

How can researchers differentiate between FABP7 and other FABP family members in experimental systems?

Differentiating between FABP7 and other FABP family members requires antibodies with validated specificity. High-quality FABP7 antibodies, such as those available commercially, demonstrate less than 5% cross-reactivity with other FABP family members (FABP1, FABP2, FABP3, etc.) in direct ELISAs . This specificity is critical given the structural similarities between FABP family proteins. When designing experiments to distinguish FABP7 from other family members, researchers should include appropriate controls: tissues known to express high levels of FABP7 (e.g., developing brain) as positive controls and, ideally, tissues from FABP7-knockout animals as definitive negative controls . For Western blot applications, the molecular weight of FABP7 (approximately 15-18 kDa) can help distinguish it from other family members, though careful interpretation is necessary due to similar molecular weights across the FABP family .

How does FABP7 expression change following CNS injury and what are the implications for neural repair?

FABP7 expression undergoes significant changes following CNS injury, with important implications for neural repair processes. In spinal cord injury models, Western blot analyses demonstrate that FABP7 is substantially upregulated after injury, reaching maximum expression at 14 days post-injury . This temporal pattern suggests involvement in the subacute to chronic phases of the injury response. Immunohistochemical analyses reveal that FABP7 is predominantly expressed in reactive astrocytes following injury, with increased co-localization of FABP7 and GFAP compared to uninjured controls . Functional studies using FABP7-knockout models provide evidence for FABP7's neuroprotective roles, as these animals demonstrate significantly reduced neuronal survival at lesion centers and caudal regions following spinal cord injury . This protective effect appears to be mediated through astrocyte functions, as FABP7 has been implicated in astrocyte proliferation and fatty acid metabolism, particularly the incorporation of omega-3 fatty acids which possess neuroprotective properties .

What is the role of FABP7 in glial cell proliferation and how can researchers investigate this function?

FABP7 plays a significant role in glial cell proliferation, particularly in reactive astrocytes following CNS injury. Research using knockout models has demonstrated that FABP7-deficient mice show decreased numbers of reactive astrocytes and BrdU-positive (proliferating) astrocytes compared to wild-type animals following cortical stab injury . This finding is further supported by in vitro studies showing that primary cultured astrocytes from FABP7-knockout mice exhibit significantly reduced proliferation rates compared to wild-type astrocytes . To investigate this function, researchers can employ multiple approaches: (1) BrdU incorporation assays combined with FABP7 and GFAP immunostaining to identify proliferating astrocytes; (2) comparative analyses between wild-type and FABP7-knockout animals following CNS injury; (3) in vitro proliferation assays using primary astrocyte cultures; and (4) molecular analyses of cell cycle regulatory pathways in the presence or absence of FABP7 . These approaches collectively provide insights into how FABP7 regulates astrocyte proliferation during reactive gliosis.

How can FABP7 antibodies be used to study the relationship between fatty acid metabolism and neural function?

FABP7 antibodies provide valuable tools for investigating the relationship between fatty acid metabolism and neural function. As a fatty acid binding protein, FABP7 plays critical roles in the uptake, transport, and metabolism of fatty acids, particularly omega-3 fatty acids which are essential for neural development and function . Research has demonstrated that primary astrocytes from FABP7-knockout mice show significantly decreased omega-3 fatty acid incorporation compared to wild-type astrocytes . To study this relationship, researchers can use FABP7 antibodies for: (1) co-immunoprecipitation experiments to identify fatty acid species bound to FABP7 in different neural cell populations; (2) immunohistochemical analyses to correlate FABP7 expression with markers of fatty acid metabolism; (3) Western blotting to compare FABP7 expression levels across different brain regions with known differences in fatty acid composition; and (4) dual-labeling approaches to examine the co-localization of FABP7 with enzymes involved in fatty acid metabolism . These approaches help elucidate how FABP7-mediated fatty acid handling contributes to neural development, function, and response to injury.

What controls should be included when using FABP7 antibodies for immunohistochemistry and Western blotting?

Proper experimental design for FABP7 antibody applications requires rigorous controls to ensure specificity and reliability of results. For immunohistochemistry applications, researchers should include: (1) primary antibody omission controls to assess non-specific binding of detection systems; (2) isotype controls to evaluate background from primary antibodies; (3) tissues known to express high levels of FABP7 (e.g., developing brain) as positive controls; and (4) ideally, tissues from FABP7-knockout animals as definitive negative controls . For Western blotting, critical controls include: (1) molecular weight markers to confirm the expected 15-18 kDa band for FABP7; (2) positive control lysates from FABP7-expressing tissues like cerebellum or hippocampus; (3) negative control lysates from tissues with minimal FABP7 expression; and (4) loading controls such as β-actin or GAPDH for normalization purposes . When using HRP-conjugated FABP7 antibodies, additional controls should verify the absence of non-specific signals due to direct enzyme conjugation.

How can researchers design experiments to investigate FABP7's role in neuroprotection after spinal cord injury?

Investigating FABP7's neuroprotective role after spinal cord injury requires a comprehensive experimental design addressing multiple aspects of the injury response. Based on evidence suggesting FABP7 may protect neurons after spinal cord injury, researchers should design experiments with the following components: (1) Temporal expression analysis using FABP7 antibodies to characterize expression patterns at multiple time points post-injury (3, 7, 10, 14, and 28 days have been validated) ; (2) Comparative studies between wild-type and FABP7-knockout animals, focusing on neuronal survival in ventral horns using NeuN immunostaining ; (3) Functional assessments of locomotor recovery to correlate molecular findings with behavioral outcomes; (4) Mechanistic investigations examining reactive astrogliosis (GFAP immunoreactivity), inflammatory responses, and fatty acid metabolism; and (5) Rescue experiments testing whether exogenous administration of fatty acids can ameliorate deficits in FABP7-knockout animals . This comprehensive approach will provide insights into how FABP7 contributes to neuroprotection following spinal cord injury.

What experimental approaches can differentiate between FABP7's role in fatty acid transport versus its signaling functions?

Distinguishing between FABP7's fatty acid transport functions and potential signaling roles requires sophisticated experimental approaches. Researchers can design studies using the following methods: (1) Subcellular localization studies using FABP7 antibodies to track protein distribution between cytoplasmic and nuclear compartments, as nuclear localization may indicate signaling functions; (2) Protein-protein interaction studies through co-immunoprecipitation with FABP7 antibodies followed by mass spectrometry to identify binding partners beyond fatty acids; (3) Fatty acid binding assays to assess FABP7's binding capacity for different fatty acids; (4) Transcriptional profiling comparing gene expression patterns in wild-type versus FABP7-knockout cells to identify signaling pathways affected by FABP7; and (5) Metabolomics approaches to analyze changes in fatty acid metabolism in the presence or absence of FABP7 . Complementary use of these approaches can help delineate FABP7's dual roles in fatty acid transport and potential signaling functions in different neural cell populations.

What are common challenges when using FABP7 antibodies and how can they be addressed?

Researchers working with FABP7 antibodies may encounter several technical challenges that require specific troubleshooting approaches. For Western blotting applications, difficulties may include weak signal detection, which can be addressed by increasing protein loading (typically 0.2 mg/mL for brain tissue lysates) or optimizing antibody concentration (50 μg/mL has been validated) . For immunohistochemistry, common issues include high background staining, which can be minimized through thorough peroxidase quenching, extended blocking steps, and optimal antibody dilution (5 μg/mL has been validated for paraffin sections) . Another challenge is variable staining patterns across different experimental conditions, which can be standardized by maintaining consistent fixation times, antigen retrieval methods, and incubation conditions. When using HRP-conjugated antibodies specifically, researchers should be vigilant about endogenous peroxidase activity in certain tissues, particularly those with high erythrocyte content, and incorporate appropriate quenching steps to minimize non-specific signal .

How can researchers optimize detection of low FABP7 expression levels in certain cell populations?

Detecting low-level FABP7 expression requires optimization strategies that enhance sensitivity while maintaining specificity. For Western blot applications, researchers can employ enhanced chemiluminescence (ECL) detection systems with extended exposure times, though care must be taken to avoid overexposure of strong signals . Sample enrichment through subcellular fractionation or immunoprecipitation may concentrate FABP7 for more reliable detection. For immunohistochemistry, signal amplification systems such as tyramide signal amplification (TSA) can significantly increase detection sensitivity for low-abundance targets. When using HRP-conjugated FABP7 antibodies, catalyzed reporter deposition techniques can amplify signal without increasing background. For both applications, reducing the volume of dilution buffer (maintaining the same antibody concentration) and extending incubation times (overnight at 4°C for primary antibodies) can enhance detection of low expression levels . These approaches have proven effective for detecting FABP7 in tissues where expression levels vary significantly between cell populations, such as in normal versus injured neural tissue .

How can researchers quantify changes in FABP7 expression across different experimental conditions?

Accurate quantification of FABP7 expression changes requires methodological rigor and appropriate analytical approaches. For Western blot quantification, densitometric analysis should be performed on bands at the expected molecular weight (15-18 kDa), with normalization to loading controls such as β-actin or GAPDH . Multiple biological replicates (minimum n=3) are essential for statistical validity, as demonstrated in studies of FABP7 expression following spinal cord injury . For immunohistochemical quantification, several approaches are validated: (1) cell counting of FABP7-positive cells within defined anatomical regions; (2) co-localization analysis to determine the percentage of specific cell types (e.g., GFAP+ astrocytes) expressing FABP7; and (3) semi-quantitative measurement of immunoreactivity intensity using standardized image acquisition parameters and analysis software . When comparing experimental conditions such as injured versus uninjured tissue, consistent tissue processing, antibody concentrations, and development times are critical for valid comparisons. Statistical approaches should account for the distribution characteristics of the data, with appropriate tests for determining significance of observed changes .