sp9 Antibody

What is SP9 and why is it significant in neurodevelopmental research?

SP9 is a zinc finger transcription factor that plays a critical role in limb development and neurogenesis. It positively regulates FGF8 expression in the apical ectodermal ridge (AER) and contributes to limb outgrowth in embryos . In the central nervous system, SP9 is widely expressed in the lateral ganglionic eminence (LGE), medial ganglionic eminence (MGE), and caudal ganglionic eminence (CGE) . Significantly, SP9 is expressed in LGE progenitors that generate nearly all striatal medium-sized spiny neurons (MSNs) and its expression is maintained specifically in postmitotic striatopallidal MSNs, but not in striatonigral MSNs .

Research has demonstrated that SP9 is crucial for:

• Generation and differentiation of striatal neurons

• Survival of striatopallidal MSNs through a Bax-dependent mechanism

• Proper development of the striatum

• Regulation of genes like Adora2a, P2ry1, Gpr6, and Grik3 in the LGE and striatum

What are the optimal applications for SP9 antibodies in experimental research?

Based on validation data across multiple sources, SP9 antibodies have been successfully employed in several applications:

For neurological studies, IHC-P has been particularly useful in analyzing SP9 expression in human cerebral cortex, where strong nuclear positivity is observed in neurons . The antibodies show localization to both nucleus and mitochondria in human A549 cells when used for ICC/IF applications .

How should SP9 antibodies be validated for developmental neuroscience experiments?

A comprehensive validation approach should include:

• Specificity testing: Verify specificity on protein arrays containing target protein plus non-specific proteins (many commercial SP9 antibodies are verified against 383+ other proteins)

• Knockout/knockdown controls: Utilize SP9 knockout models as negative controls. The search results mention several SP9 mutant alleles including SP9-LacZ null mice where SP9 protein and RNA are not detectable

• Expression pattern verification: Compare antibody staining with known SP9 expression patterns:

  • SP9 is expressed in SVZ and mantle zone of LGE, MGE and CGE at E13.5

  • In postmitotic neurons, SP9 is specifically expressed in striatopallidal neurons but not striatonigral neurons

  • In adult striatum, virtually all Drd2+ cells express SP9, while Drd1+ cells do not

• Cross-reactivity assessment: Test antibody reactivity across species. Many SP9 antibodies show 100% sequence identity with mouse and rat SP9 in the immunogen region

What are the critical parameters for optimizing SP9 antibody-based immunohistochemistry?

For optimal IHC results with SP9 antibodies:

• Fixation: Paraffin-embedded tissues are widely used in published SP9 studies

• Antigen retrieval: Heat-mediated antigen retrieval with citrate buffer pH 6 is crucial before IHC staining protocols

• Antibody concentration: Titration between 1:50-1:200 dilution is recommended for paraffin sections

• Incubation conditions: Temperature and duration should be optimized based on tissue type and fixation method

• Detection systems: Both chromogenic and fluorescent detection systems have been validated with SP9 antibodies

• Controls: Include positive controls (cerebral cortex shows strong nuclear positivity) and negative controls (stomach glandular cells show no positivity as expected)

How can SP9 antibodies be utilized to study neurogenesis in the telencephalon?

SP9 antibodies can be strategically employed in fate-mapping and developmental studies:

• Lineage tracing experiments: Using SP9-Cre knockin mice crossed with reporter lines (e.g., Rosa-YFP), researchers have demonstrated that SP9+ progenitors generate:

  • 96% of cortical interneurons (including PV+, SST+, CR+, NPY+ and VIP+ subtypes)

  • Virtually all striatal interneurons (PV+, SST+, CR+ and ChAT+)

  • Almost all Foxp1+ medium spiny neurons in the striatum

• Temporal expression analysis: SP9 expression can be tracked throughout development using timed immunostaining:

  • Detectable in ganglionic eminences as early as E10.5

  • Expressed in SVZ and mantle zone at E13.5

  • Maintained in adult striatum at lower levels, with specificity for Drd2+ cells

• Proliferation studies: Combine SP9 antibodies with cell proliferation markers (e.g., Ki67, BrdU) to identify dividing progenitors:

  • A subset of Sp9+ cells in the SVZ express both Ascl1 and Ki67

  • BrdU pulse-labeling experiments show that SP9 regulates cell proliferation in the LGE SVZ

What approaches can resolve contradictory SP9 antibody staining patterns in different neural tissues?

When faced with discrepant staining patterns:

• Epitope mapping: Different antibodies target different regions of SP9 protein:

  • Some antibodies target amino acids 400 to C-terminus

  • Others target amino acids 187-237

  • Some target the sequence SKHIKTHNGGGGGKKGSDSDTDASNLETPRSESPDLILHDSGVSA

• Isoform specificity: Check if antibodies detect all SP9 isoforms or are isoform-specific

• Cross-validation: Use multiple antibodies targeting different epitopes and compare results

• Complementary approaches: Validate antibody staining with RNA in situ hybridization:

  • SP9 in situ RNA hybridization confirms antibody patterns in ganglionic eminences

  • For striatopallidal neurons, correlate SP9 staining with Drd2, Penk, Gpr6, Adora2a, and Ptprm expression

How can SP9 antibodies be integrated with advanced imaging techniques for single-cell analysis?

For high-resolution, single-cell analysis of SP9 expression:

• Super-resolution microscopy: Techniques like STORM or STED can resolve SP9 subcellular localization beyond the diffraction limit, revealing nuclear distribution patterns

• Expansion microscopy: Physical expansion of specimens allows visualization of SP9 protein distribution in cellular compartments using standard confocal microscopy

• Multiplexed immunofluorescence: Combining SP9 antibodies with other markers:

  • Use SP9 with Ascl1 and Ki67 to identify proliferating progenitors

  • Combine with Foxp1 and Drd2-GFP to identify striatopallidal MSNs

  • Co-stain with Drd1-GFP to distinguish from striatonigral MSNs

• Quantitative image analysis: Develop algorithms to quantify:

  • Nuclear SP9 intensity across development

  • Co-localization coefficients with other transcription factors

  • Spatial distribution patterns in the developing striatum

How can SP9 antibodies be employed in ChIP assays to study SP9 transcriptional targets?

For effective chromatin immunoprecipitation with SP9 antibodies:

• Antibody selection: Choose antibodies validated for immunoprecipitation applications

  • Ensure the epitope is accessible in the chromatin context

  • Verify antibody works in IP before proceeding to ChIP

• Cross-linking optimization: Determine optimal formaldehyde concentration and cross-linking time for SP9

• Sonication parameters: Optimize sonication conditions to generate chromatin fragments of 200-500 bp

• Target validation: Based on published research, initial ChIP-qPCR should focus on:

  • SP9 regulatory regions (Ascl1 directly binds the SP9 promoter )

  • Putative SP9 targets including Adora2a, P2ry1, Gpr6, and Grik3 regulatory regions

• Controls: Include:

  • Input chromatin

  • Non-specific IgG antibody

  • Positive control regions (known SP9 binding sites)

  • Negative control regions (non-binding regions)

What strategies can optimize protein-protein interaction studies using SP9 antibodies?

For investigating SP9 protein complexes:

• Co-immunoprecipitation (Co-IP) approach:

  • Optimize lysis buffers to preserve native SP9 protein complexes

  • Use antibodies targeting different SP9 epitopes to avoid disrupting specific interactions

  • Consider crosslinking strategies for transient interactions

• Proximity ligation assay (PLA) workflow:

  • Combine SP9 antibodies with antibodies against suspected interaction partners

  • PLA signals indicate proteins are within 40 nm of each other

  • Effective for visualizing interactions in situ in tissue sections

• FRET/FLIM analysis:

  • Label SP9 antibodies and potential partners with appropriate fluorophore pairs

  • Detect energy transfer indicating close molecular proximity

• Potential interaction partners to investigate based on research findings:

  • Ascl1 (upstream regulator of SP9 )

  • Components of the Bax-dependent apoptotic machinery (SP9 regulation of cell survival )

  • Transcriptional co-factors involved in regulating Adora2a, P2ry1, Gpr6, and Grik3

How can SP9 antibodies contribute to understanding neurological disorders?

SP9 research has significant implications for several neurological conditions:

• Movement disorders: Given SP9's critical role in striatopallidal MSN development, SP9 antibodies can be used to:

  • Assess striatopallidal neuron integrity in Parkinson's disease models

  • Evaluate striatal neuron populations in Huntington's disease

  • Study developmental origins of dystonia and other basal ganglia disorders

• Neurodevelopmental disorders: SP9's role in telencephalon development suggests applications in:

  • Analyzing cortical interneuron populations in autism spectrum disorders

  • Investigating developmental disruptions in schizophrenia

  • Studying aberrant neuronal migration in cortical malformations

• Methodological approach:

  • Quantify SP9+ neurons in postmortem brain tissue

  • Compare SP9 expression patterns in control vs. disease models

  • Correlate SP9+ cell counts with clinical measures of disease severity

What methodological considerations are important when using SP9 antibodies in human postmortem tissue studies?

Working with human postmortem tissue presents specific challenges:

• Fixation effects: Postmortem interval and fixation method significantly impact antibody performance

  • Test multiple antigen retrieval methods (citrate buffer pH 6 is recommended but other buffers may be needed )

  • Optimize antibody concentration for each case based on fixation quality

• Autofluorescence management:

  • Use Sudan Black B or TrueBlack to quench lipofuscin autofluorescence

  • Consider spectral imaging to separate antibody signal from tissue autofluorescence

• Validated controls:

  • Human cerebral cortex shows strong nuclear positivity in neurons

  • Human stomach glandular cells show no positivity (negative control)

  • Human fallopian tube tissue can be used for antibody validation

• Data analysis adaptations:

  • Account for age-related changes in SP9 expression

  • Consider co-morbidities that might affect neuronal populations

  • Use stereological methods for unbiased quantification