PDE1A Antibody, FITC conjugated

What is PDE1A and what are its primary functions in cellular signaling?

PDE1A (Phosphodiesterase 1A) is a calcium/calmodulin-dependent enzyme that hydrolyzes cyclic nucleotides (cAMP and cGMP) to their respective nucleoside 5'-monophosphates, playing a crucial role in signal transduction. It has a dual specificity for both cAMP and cGMP, though it demonstrates a higher affinity for cGMP. As part of the cyclic nucleotide phosphodiesterase (PDEs) family, PDE1A regulates intracellular cyclic nucleotide concentrations, which are key second messengers in many important physiological processes. The protein exists in multiple isoforms (including PDE1A1, PDE1A3, PDE1A5) produced by alternative splicing, with molecular weights ranging from approximately 57-63 kDa .

What tissue expression patterns are observed for PDE1A in different species?

PDE1A expression patterns show both conservation and variation across species. mRNA studies demonstrate that PDE1A is detected at nearly equivalent levels in human, rat, and mouse hearts. In contrast, PDE1C (another family member) is primarily detected in human and mouse hearts, while PDE1B shows weak expression in cardiac tissue across species .

PDE1A protein is consistently detected in:

• Brain tissue (human, mouse, rat)

• Cardiac tissue (human, mouse, rat)

• Human gliomas tissue

Western blot analysis confirms PDE1A expression in brain tissues across all three species, and immunohistochemistry verifies its presence in brain tissue and human gliomas .

How does FITC conjugation affect the application spectrum of PDE1A antibodies?

FITC (Fluorescein isothiocyanate) conjugation expands the application range of PDE1A antibodies by enabling direct fluorescent detection without secondary antibodies. This modification particularly enhances applications in:

• Immunofluorescence (IF) - Allowing direct visualization of PDE1A localization in cells and tissues

• Flow cytometry - Enabling quantitative analysis of PDE1A expression in cell populations

• Immunocytochemistry (ICC) - Providing direct visualization in cultured cells

The FITC-conjugated antibody is typically supplied in a stabilization buffer containing preservatives (0.03% Proclin 300) and 50% glycerol in PBS (pH 7.4) . This formulation maintains antibody stability while preserving the fluorescent properties of FITC. Researchers should note that FITC has an excitation maximum at approximately 495 nm and an emission maximum around 519 nm, making it compatible with standard FITC filter sets on fluorescence microscopes .

What are the optimal dilution ratios and sample preparation methods for different experimental applications?

Optimal dilution ratios vary significantly depending on the experimental application and sample type. Based on compiled data from multiple sources, the following recommendations can serve as starting points:

For all applications, it is recommended to titrate the antibody in each testing system to obtain optimal results, as performance can be sample-dependent . When working with FITC-conjugated antibodies specifically, samples should be protected from light during incubation to prevent photobleaching of the fluorophore .

How should PDE1A antibody be stored and handled to maintain optimal reactivity?

Proper storage and handling of PDE1A antibodies, particularly FITC-conjugated versions, is critical for maintaining reactivity and fluorescence intensity. Based on manufacturer recommendations:

Long-term storage:

• Store at -20°C for FITC-conjugated antibodies

• Some suppliers recommend -80°C for unconjugated versions

• Avoid repeated freeze-thaw cycles that can degrade both antibody function and FITC fluorescence

Working solution preparation:

• Thaw aliquots at room temperature or 4°C

• For 50 μL volume products with concentration around 0.59 μg/μL, dilute in appropriate buffers immediately before use

• For some formulations (particularly those in 50% glycerol), aliquoting may be unnecessary for -20°C storage

Light exposure:

• Minimize exposure to light during all handling steps

• Work in reduced ambient lighting when preparing FITC-conjugated antibody dilutions

• Store in amber tubes or wrapped in aluminum foil

Stability period:

• Most preparations remain stable for one year after shipment when stored properly

• Working dilutions should be prepared fresh and used within 24 hours

What validation methods confirm the specificity of PDE1A antibodies in experimental systems?

Validating PDE1A antibody specificity is essential for generating reliable experimental data. Multiple complementary approaches should be employed:

• Western blot analysis with positive controls:

  • Human, mouse, and rat brain tissues show consistent detection of the expected 61 kDa band

  • Multiple antibodies validated against recombinant PDE1A protein show similar patterns

• Genetic validation approaches:

  • siRNA or shRNA knockdown experiments demonstrate specificity:

    • PDE1A siRNA targeting N-terminal sequence significantly down-regulates PDE1A protein levels

    • PDE1A shRNA targeting C-terminal sequence confirms specificity

    • Negative controls using non-targeting siRNA show no effect on PDE1A expression

    • Importantly, PDE1A siRNA does not alter expression of other PDE isoforms (PDE1C, PDE5A)

• Immunogen verification:

  • Antibodies raised against specific PDE1A recombinant proteins (e.g., amino acids 1-219 or region within 1-50)

  • Synthetic peptide immunogens corresponding to unique amino acid sequences within the protein

• Cross-reactivity testing:

  • Verification that antibodies do not label other PDE family members

  • Confirmation that antibodies label all relevant PDE1A variants (PDE1A1, PDE1A3, PDE1A5)

• Immunohistochemical pattern analysis:

  • Characteristic staining patterns in tissues with known PDE1A expression

  • Comparison of staining patterns between normal and pathological tissues (e.g., increased PDE1A staining in hypertrophic hearts)

How does PDE1A expression change in pathological cardiac hypertrophy models?

PDE1A expression undergoes significant upregulation in multiple pathological cardiac hypertrophy models across different species. Comprehensive immunohistochemical analyses reveal consistent patterns:

• Transverse Aortic Constriction (TAC) model:

  • PDE1A staining is significantly increased in the myocardium of TAC-induced hypertrophic hearts compared to sham controls

  • The increase is prominently detected in cardiomyocytes, though not exclusively limited to them

• Chronic Isoproterenol (ISO) infusion model:

  • Mouse hearts with chronic ISO infusion show similar upregulation of PDE1A compared to controls

  • This model represents sympathetic overstimulation-induced hypertrophy

• Angiotensin II (Ang II) infusion model:

  • Rat hearts with Ang II infusion demonstrate comparable increases in PDE1A expression

  • This represents a renin-angiotensin system-induced hypertrophy model

These findings across multiple hypertrophy models suggest that PDE1A upregulation is a common response in pathological cardiac remodeling, regardless of the initiating stimulus. The functional significance of this upregulation has been demonstrated through loss-of-function studies where:

• PDE1A gene silencing via siRNA significantly abrogated phenylephrine (PE)-mediated increases in protein synthesis

• PDE1A knockdown reduced total myocyte surface area compared to controls in neonatal rat ventricular myocytes (NRVM)

• PDE1A inhibition correlated with reduced mRNA levels of hypertrophic markers, including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)

These findings position PDE1A as a potential therapeutic target in pathological cardiac hypertrophy.

What is the functional role of PDE1A in regulating cGMP/PKG signaling in cardiomyocytes?

PDE1A plays a critical role in regulating cGMP/PKG signaling in cardiomyocytes, particularly in the context of pathological hypertrophy. The enzyme demonstrates preferential hydrolysis of cGMP over cAMP in vascular smooth muscle cells (VSMCs) and similar preferences in cardiac tissues .

Functional studies reveal:

• cGMP/PKG pathway modulation:

  • PDE1A regulates cardiomyocyte hypertrophy by modulating cGMP/PKG signaling

  • This effect was demonstrated using various loss-of-function strategies in both neonatal rat ventricular myocytes (NRVM) and adult rat ventricular myocytes (ARVM)

  • The anti-hypertrophic effects of PDE1 inhibition are dependent on the cGMP/PKG pathway

• Calcium/calmodulin regulation:

  • As a Ca²⁺/calmodulin-stimulated phosphodiesterase, PDE1A activity increases in response to elevated intracellular calcium

  • This creates a feedback mechanism where calcium signaling (often elevated in hypertrophy) enhances PDE1A activity, which then reduces cGMP levels

  • The reduction in cGMP attenuates protein kinase G (PKG) activity, a known negative regulator of hypertrophic signaling

• Therapeutic potential:

  • The PDE1-selective inhibitor IC86340 reduced myocyte hypertrophy in an isoproterenol-induced hypertrophy mouse model

  • This effect correlated with reduced expression of hypertrophic markers (ANP, BNP)

  • Perinuclear ANP immunostaining showed that phenylephrine (PE) markedly stimulated the percentage of ANP/actinin-positive cells compared to controls, while IC86340 treatment abrogated these effects

This body of evidence establishes PDE1A as a critical regulator of cardiomyocyte hypertrophy through its effects on cGMP/PKG signaling, positioning it as a potential therapeutic target in pathological cardiac remodeling.

How can FITC-conjugated PDE1A antibodies be utilized in multiplexed immunofluorescence studies?

FITC-conjugated PDE1A antibodies offer significant advantages in multiplexed immunofluorescence studies, allowing researchers to simultaneously visualize PDE1A alongside other proteins of interest. Optimal multiplexed protocols leverage FITC's spectral properties while avoiding cross-reactivity and signal interference:

• Compatible fluorophore combinations:

  • FITC (excitation ~495nm, emission ~519nm) pairs well with:

    • TRITC/Rhodamine (red)

    • Cy5 (far-red)

    • DAPI (blue, for nuclear counterstaining)

  • Avoid fluorophores with significant spectral overlap such as GFP or Alexa Fluor 488

• Experimental design considerations:

  • Primary antibody selection: Choose primary antibodies from different host species (e.g., FITC-conjugated rabbit anti-PDE1A combined with mouse antibodies against other targets)

  • Sequential staining: For same-species primary antibodies, use sequential staining with complete blocking between steps

  • Controls: Include single-stained controls to assess bleed-through and autofluorescence

• PDE1A localization studies:

  • Co-localization with subcellular markers: FITC-PDE1A antibodies can be combined with markers for:

    • Perinuclear regions (where ANP accumulates during hypertrophy)

    • Sarcomeric structures (using α-actinin antibodies)

    • Membrane components (using caveolin or other membrane markers)

  • This approach has been successfully used to demonstrate PDE1A expression changes in cardiomyocytes during hypertrophy

• Quantitative analysis techniques:

  • Co-localization coefficients (Pearson's, Manders') can quantify spatial relationships between PDE1A and other proteins

  • Signal intensity measurements across experimental conditions can provide semi-quantitative data on expression changes

  • 3D reconstruction from confocal z-stacks can reveal spatial distribution patterns within complex tissues

When working with FITC-conjugated antibodies in multiplexed studies, researchers should be particularly mindful of fixation methods, as different fixatives may differentially affect epitope accessibility and fluorophore stability across antibodies .

What are common technical challenges when using FITC-conjugated PDE1A antibodies, and how can they be addressed?

Working with FITC-conjugated PDE1A antibodies presents several technical challenges that can impact experimental outcomes. The following table outlines common issues and their solutions:

For particularly challenging applications, consider these advanced troubleshooting approaches:

• For weak signals in brain tissue:

  • Extend primary antibody incubation time to overnight at 4°C

  • Increase antibody concentration (but monitor background)

  • Use signal amplification systems like tyramide signal amplification (TSA)

• For high background in cardiac tissue:

  • Implement additional blocking steps using animal serum from the same species as the secondary antibody

  • Include 0.1-0.3% Triton X-100 in blocking solutions to reduce non-specific binding

  • Perform extensive washing steps (5-6 washes of 5 minutes each)

• For tissues with high autofluorescence:

  • Treat sections with Sudan Black B (0.1-0.3% in 70% ethanol) to quench lipofuscin autofluorescence

  • Consider spectral unmixing during image acquisition on confocal microscopes

How can researchers quantitatively analyze PDE1A expression changes in hypertrophic cardiomyocyte models?

Quantitative analysis of PDE1A expression changes in hypertrophic cardiomyocyte models requires rigorous methodological approaches across multiple platforms. Based on published research protocols:

• Western blot quantification:

  • Prepare standardized lysates from control and hypertrophic samples (recommended dilution 1:1000-1:6000)

  • Include loading controls (GAPDH, β-actin) and normalize PDE1A band intensity

  • Use digital imaging software (ImageJ, Bio-Rad Image Lab) for densitometric analysis

  • Present data as fold-change relative to control conditions

  • Statistical significance should be determined using appropriate tests (typically t-test or ANOVA)

• Immunohistochemical analysis:

  • Capture standardized images from control and hypertrophic heart sections (recommended antibody dilution 1:50-1:500)

  • Analyze using:

    • Intensity scoring (0-3+ scale)

    • Percentage of positive cells

    • Histological scoring (H-score = % cells × intensity)

  • For cardiomyocyte-specific analysis, co-stain with sarcomeric markers (α-actinin)

  • Blinded scoring by multiple observers improves reliability

• mRNA expression analysis:

  • Complement protein studies with qRT-PCR for PDE1A transcripts

  • Design primers specific to PDE1A variants

  • Normalize to stable reference genes (validated in hypertrophy models)

  • Compare with hypertrophic markers (ANP, BNP) to correlate PDE1A changes with hypertrophy severity

• Functional assays:

  • Measure PDE1A enzymatic activity using cyclic nucleotide hydrolysis assays

  • Correlate activity with protein expression levels

  • Assess cGMP levels in control vs. hypertrophic samples to demonstrate functional impact

For mechanistic studies, the following findings provide context for interpretation:

• PDE1A expression increased significantly in multiple hypertrophy models (TAC, ISO infusion, Ang II infusion)

• PDE1A knockdown (via siRNA or shRNA) significantly reduced protein synthesis and myocyte surface area in hypertrophic conditions

• Changes in PDE1A correlated with classical hypertrophy markers (ANP, BNP)

What strategies help distinguish between different PDE1A splice variants in experimental samples?

Distinguishing between PDE1A splice variants requires specialized approaches that target their unique structural and functional characteristics. PDE1A exists in multiple isoforms (including PDE1A1, PDE1A3, PDE1A5) with molecular weights ranging from approximately 57-63 kDa . To differentiate between these variants:

• Isoform-specific antibodies:

  • Some antibodies recognize all PDE1A variants (e.g., those targeting common domains)

  • For variant specificity, use antibodies raised against unique N-terminal sequences

• RT-PCR and qPCR strategies:

  • Design primers flanking splice junctions unique to specific variants

  • Nest forward primers in unique exons of each variant

  • Expected product sizes:

    Variant	Approximate Size	Key Distinguishing Features
    PDE1A1	Variable	Contains N-terminal regulatory domain
    PDE1A2	Variable	Lacks specific inhibitory sequence
    PDE1A3-5	Variable	Different N-terminal regions

  • Verify amplicon identity through sequencing

• High-resolution protein separation:

  • Employ 2D gel electrophoresis to separate variants based on both molecular weight and isoelectric point

  • Use gradient gels (7.5-15%) to enhance resolution in the 57-63 kDa range

  • Follow with Western blotting using pan-PDE1A antibodies

• Mass spectrometry approaches:

  • Perform tryptic digestion followed by LC-MS/MS

  • Identify variant-specific peptide sequences

  • Quantify relative abundance of variants using label-free or isotope-labeled techniques

• Functional discrimination:

  • Exploit differences in Ca²⁺/calmodulin sensitivity between variants

  • Measure cyclic nucleotide hydrolysis kinetics at varying calcium concentrations

  • Compare substrate preferences (cAMP vs. cGMP) as variants may differ in their selectivity

When analyzing cardiac samples specifically, researchers should note that PDE1A (rather than PDE1C) expression appears more conserved in rodent hearts compared to human hearts, which may impact translational research interpretation .

How is FITC-conjugated PDE1A antibody being utilized in neurodegenerative disease research?

FITC-conjugated PDE1A antibodies are emerging as valuable tools in neurodegenerative disease research, building on established evidence of PDE1A expression in brain tissues across species. Current applications leverage the direct visualization capabilities of FITC conjugation:

• Alzheimer's disease investigations:

  • PDE1A is being studied in relation to cGMP signaling pathways, which are increasingly implicated in Alzheimer's pathophysiology

  • FITC-conjugated antibodies enable co-localization studies with Aβ plaques and tau tangles

  • Expression pattern changes in disease progression can be tracked using standardized immunofluorescence protocols (typically starting at 1:50-1:200 dilution)

  • Human gliomas tissue has been successfully labeled with PDE1A antibodies, suggesting applicability to other CNS pathologies

• Cerebrovascular dysfunction studies:

  • PDE1A's role in vascular smooth muscle cells and its preferential cGMP hydrolysis activity position it as a potential mediator in cerebrovascular pathology

  • FITC-conjugated antibodies facilitate visualization of PDE1A in the neurovascular unit

  • Co-staining with endothelial and smooth muscle markers helps elucidate PDE1A's role in the blood-brain barrier

• Synaptic plasticity research:

  • cGMP/PKG signaling influences synaptic plasticity and memory formation

  • PDE1A regulation of these pathways can be visualized using FITC-conjugated antibodies in:

    • Hippocampal slice cultures

    • Primary neuronal cultures

    • Animal models of learning and memory

• High-resolution microscopy applications:

  • Super-resolution techniques (STED, STORM) are being combined with FITC-conjugated PDE1A antibodies to reveal subcellular localization

  • This approach helps determine whether PDE1A occupies specific microdomains within neurons that influence local cyclic nucleotide signaling

The strong expression of PDE1A in brain tissue across species (human, mouse, rat) makes it a particularly attractive target for translational neuroscience research .

What advances in PDE1A inhibitor development might impact future research applications of FITC-conjugated antibodies?

The development of novel PDE1A inhibitors represents a significant frontier that will likely expand applications for FITC-conjugated PDE1A antibodies in both basic research and therapeutic development:

• Target engagement studies:

  • FITC-conjugated PDE1A antibodies enable direct visualization of inhibitor-target interactions through:

    • Competitive binding assays measuring displacement of antibodies by inhibitors

    • Conformational changes in PDE1A upon inhibitor binding detected by altered epitope accessibility

  • These approaches help validate the mechanism of action for compounds like IC86340 (a PDE1-selective inhibitor) that has shown efficacy in reducing myocyte hypertrophy

• Inhibitor specificity assessment:

  • FITC-conjugated antibodies facilitate screening of inhibitor effects across tissues expressing different PDE1A splice variants

  • Comparative analysis can be performed using standardized immunofluorescence protocols (1:200 dilution recommended as starting point)

  • This helps address whether inhibitors show preferential activity against specific variants

• Pharmacodynamic biomarker development:

  • FITC-PDE1A immunofluorescence could serve as a companion diagnostic tool to assess:

    • Target expression levels prior to inhibitor treatment

    • Changes in PDE1A expression/localization in response to inhibitor therapy

    • Correlation between expression patterns and treatment outcomes

• Combined therapeutic approaches:

  • Research into synergistic effects of PDE1A inhibitors with other interventions can utilize FITC-conjugated antibodies to:

    • Track changes in signaling pathway components (e.g., PKG activation)

    • Visualize downstream effects on hypertrophic markers like perinuclear ANP accumulation

    • Monitor cellular remodeling through co-staining with structural proteins

• Translational model development:

  • FITC-conjugated antibodies that demonstrate cross-reactivity with human, mouse, and rat PDE1A enable:

    • Validation of animal models for inhibitor testing

    • Assessment of target conservation across species

    • Development of humanized models with accurate PDE1A expression patterns

The PDE1-selective inhibitor IC86340, which has demonstrated efficacy in reducing cardiomyocyte hypertrophy and hypertrophic marker expression, exemplifies how inhibitor development drives expanded applications for immunofluorescence-based PDE1A research .

How might single-cell imaging techniques advance our understanding of PDE1A's compartmentalized signaling in complex tissues?

Single-cell imaging techniques combined with FITC-conjugated PDE1A antibodies offer transformative potential for understanding compartmentalized cyclic nucleotide signaling in complex tissues: