SFTPD Antibody, FITC conjugated

What is Surfactant Protein D (SFTPD) and why is it important in respiratory research?

Surfactant Protein D (SFTPD) is a hydrophilic collectin found primarily in the lungs that plays a crucial role in innate immunity and surfactant homeostasis. SFTPD functions to protect against microbial challenges by binding to lipid components of bacterial cell walls, facilitating rapid removal of pathogens . SFTPD is secreted and found in the extracellular matrix, making it an important biomarker for lung function and pathology. The protein is encoded by the SFTPD gene and works alongside other surfactant proteins to maintain alveolar stability by modulating surface tension at the air-liquid interface in peripheral air spaces. Research involving SFTPD is critical for understanding pulmonary diseases, inflammatory responses, and host defense mechanisms in the respiratory system.

What is FITC conjugation and what are the spectral properties of FITC-conjugated antibodies?

Fluorescein isothiocyanate (FITC) conjugation is a chemical process that covalently links the fluorescent molecule FITC to proteins, particularly antibodies, to create fluorescently labeled reagents for various applications. FITC reacts with free amino groups of proteins to form stable conjugates that can be detected using fluorescence-based techniques .

FITC has the following spectral properties:

• Absorption maximum: 495 nm

• Emission maximum: 525 nm (bright green fluorescence)

These spectral characteristics make FITC-conjugated antibodies ideal for:

• Fluorescence microscopy

• Flow cytometry

• Immunohistochemistry

• Multiplex immunofluorescence assays with minimal spectral overlap

The high quantum efficiency and stability of FITC conjugates contribute to their widespread use in research settings, though care must be taken to minimize photobleaching during extended imaging sessions .

What are the optimal storage conditions for SFTPD Antibody, FITC conjugated?

To maintain the activity and fluorescence of SFTPD Antibody, FITC conjugated reagents, the following storage conditions are recommended:

• Store at -20°C for long-term storage

• Aliquot into multiple vials to avoid repeated freeze-thaw cycles, which can significantly degrade both the antibody and the fluorophore

• Protect from light at all times to prevent photobleaching of the FITC fluorophore

• For working solutions, add 1% (w/v) BSA and 0.1% (w/v) sodium azide as preservatives

• When stored properly, most FITC-conjugated antibodies maintain activity for at least 12 months

Most commercially available SFTPD antibodies with FITC conjugation are supplied in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol, which helps maintain stability during freeze-thaw cycles .

What are the primary applications of SFTPD Antibody, FITC conjugated in immunofluorescence studies?

SFTPD Antibody, FITC conjugated is versatile in immunofluorescence applications and can be used in several formats:

• Immunohistochemistry on paraffin-embedded tissues (IHC-P): Allows visualization of SFTPD expression in fixed lung tissue sections, typically at dilutions of 1:50-200 . This application is particularly valuable for studying SFTPD distribution in normal versus diseased lung tissue.

• Immunohistochemistry on frozen sections (IHC-F): Enables detection of SFTPD in frozen tissue sections while preserving native protein conformation, typically used at dilutions of 1:50-200 .

• Immunocytochemistry (ICC): Permits detection of SFTPD in cultured cells including type II pneumocytes and other SFTPD-expressing cells, with recommended dilutions of 1:50-200 .

• Multiplex immunofluorescence: FITC's spectral properties allow it to be combined with other fluorophores (e.g., TRITC, Cy5) for simultaneous detection of multiple proteins in the same sample.

The bright green fluorescence of FITC makes it ideal for visualizing SFTPD localization in cellular and tissue contexts, providing insights into protein distribution and expression levels in both physiological and pathological states.

How can SFTPD Antibody, FITC conjugated be optimally used in flow cytometry?

Flow cytometry is a powerful application for SFTPD Antibody, FITC conjugated, allowing quantitative analysis of SFTPD expression in cell populations. For optimal results:

• Sample preparation: Single-cell suspensions should be prepared from tissues or cell cultures with minimal cell clumping

• Antibody dilution: Use at recommended dilutions (typically 1:20-100 for flow cytometry)

• Controls required:

  • Unstained cells (for autofluorescence assessment)

  • Isotype control, FITC-conjugated (to determine non-specific binding)

  • Positive control (cells known to express SFTPD)

  • Negative control (cells known to lack SFTPD expression)

• Gating strategy: Initial gating should exclude debris and dead cells, followed by analysis of SFTPD-positive populations

• Data analysis considerations:

  • Mean fluorescence intensity (MFI) provides quantitative measurement of SFTPD expression

  • Percentage of positive cells indicates the proportion of cells expressing detectable SFTPD

  • Histogram overlays can visualize shifts in SFTPD expression between experimental conditions

Flow cytometry with SFTPD Antibody, FITC conjugated is particularly valuable for studying SFTPD expression in bronchoalveolar lavage samples, lung-derived cell suspensions, and cultured cells under various experimental conditions.

What is the optimal fluorescein/protein (F/P) ratio for SFTPD antibody conjugation, and how is it determined?

The fluorescein/protein (F/P) ratio is a critical parameter in FITC antibody conjugation that significantly impacts performance. For optimal SFTPD antibody conjugation:

• Ideal F/P ratio range: For most immunofluorescence applications, an F/P ratio between 2.5-6.0 is considered optimal for FITC-conjugated antibodies

• Determination of F/P ratio: The ratio can be calculated using spectrophotometric measurements with the following formula:
  $Molar\ F/P = \frac{2.77 \times A_{495}}{A_{280} - (0.35 \times A_{495})}$

• Impact of ratio variations:

  • F/P ratios <2: May result in insufficient signal intensity

  • F/P ratios >6: Often lead to increased non-specific binding, fluorescence quenching, and altered antibody specificity

• Optimization process: Small-scale test conjugations at different molar ratios (typically 5:1, 10:1, and 20:1 FITC:antibody) should be performed to determine the optimal ratio before scaling up

The optimal F/P ratio ensures maximum sensitivity while maintaining antibody specificity and minimizing background. When properly optimized, the FITC-conjugated SFTPD antibody will provide consistent and reliable results across experimental applications.

What are the critical parameters for optimizing FITC conjugation to SFTPD antibodies?

Successful FITC conjugation to SFTPD antibodies depends on several critical parameters that must be carefully controlled:

Additional considerations include:

• Buffer composition: 0.1M carbonate-bicarbonate buffer (pH 9.0) provides optimal conditions for FITC conjugation

• FITC quality: Use high-quality, freshly prepared FITC solution within 5 minutes of preparation to prevent hydrolysis

• Purification method: Gel filtration using Sephadex G-25 effectively separates conjugated antibody from free FITC

• Post-conjugation stabilization: Adding 1% BSA and 0.1% sodium azide helps stabilize the conjugate for storage

Careful attention to these parameters ensures consistent production of high-quality SFTPD Antibody, FITC conjugated with optimal detection sensitivity and specificity.

How can researchers troubleshoot high background fluorescence when using SFTPD Antibody, FITC conjugated?

High background fluorescence is a common challenge when working with FITC-conjugated antibodies. To troubleshoot this issue:

• Potential causes and solutions:

  • Over-labeling of antibody (F/P ratio too high):

    • Use antibodies with optimal F/P ratios (2.5-6.0)

    • Purify over-labeled antibodies using DEAE Sephadex chromatography to isolate optimally labeled fractions

  • Non-specific binding:

    • Increase blocking duration (use 5% BSA or 10% normal serum from the same species as the secondary antibody)

    • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

    • Include additional washing steps with 0.05% Tween-20 in buffer

  • Autofluorescence:

    • Treat sections with 0.1% sodium borohydride or 0.3% Sudan Black B in 70% ethanol

    • Use spectral unmixing during image acquisition if available

  • Fixation-induced fluorescence:

    • Optimize fixation protocol (reduce fixation time)

    • Use freshly prepared paraformaldehyde rather than formalin

• Recommended controls:

  • Include isotype control antibody, FITC-conjugated, at the same concentration

  • Perform secondary-only controls (for indirect methods)

  • Include known negative tissues/cells

• Image acquisition adjustments:

  • Optimize exposure settings based on negative controls

  • Consider employing spectral imaging to distinguish FITC signal from autofluorescence

Reducing background fluorescence is essential for accurate interpretation of SFTPD localization and expression patterns, especially in tissues with high intrinsic autofluorescence like lung tissue.

What controls should be included when using SFTPD Antibody, FITC conjugated for research validation?

Proper experimental controls are essential for validating results obtained with SFTPD Antibody, FITC conjugated:

• Essential negative controls:

  • Isotype control: FITC-conjugated rabbit IgG (matching the host species of the SFTPD antibody) at the same concentration as the primary antibody

  • Absorption control: Pre-incubating the SFTPD Antibody, FITC conjugated with excess recombinant SFTPD protein before staining

  • Secondary-only control: For indirect detection methods

  • Untreated/unstained samples: To assess autofluorescence levels

• Positive controls:

  • Known positive tissues: Human lung tissue sections known to express SFTPD

  • Recombinant SFTPD-expressing cells: Overexpression systems providing strong positive signal

  • Western blot validation: Confirming antibody specificity via molecular weight verification

• Procedure controls:

  • Titration series: Testing multiple antibody dilutions to determine optimal signal-to-noise ratio

  • Cross-reactivity assessment: Testing the antibody on tissues from predicted reactive species (cow, sheep, rabbit)

  • Multi-method validation: Confirming findings using alternative detection methods (e.g., immunohistochemistry, western blotting)

• Quantification controls:

  • Calibration standards: For quantitative applications

  • Internal reference markers: For normalization between samples

Proper implementation of these controls ensures research validity and facilitates troubleshooting when unexpected results occur. Documentation of all control results should be maintained to support publication of findings.

How are SFTPD Antibody, FITC conjugated reagents being used in advanced microscopy techniques?

Recent advances in microscopy have expanded the applications of SFTPD Antibody, FITC conjugated beyond conventional fluorescence microscopy:

• Super-resolution microscopy:

  • Structured Illumination Microscopy (SIM): Enables visualization of SFTPD distribution at ~100 nm resolution, revealing previously undetectable subcellular localization patterns

  • Stochastic Optical Reconstruction Microscopy (STORM): Achieves 20-30 nm resolution with FITC-conjugated antibodies, allowing precise mapping of SFTPD distribution within lamellar bodies and at the cell surface

  • Stimulated Emission Depletion (STED): Provides detailed visualization of SFTPD interactions with microbial pathogens and other surfactant components

• Live-cell imaging applications:

  • Development of cell-permeable FITC-conjugated Fab fragments against SFTPD for real-time tracking of protein dynamics

  • Combination with pH-sensitive probes to monitor SFTPD trafficking in acidic cellular compartments

• Correlative Light and Electron Microscopy (CLEM):

  • Integration of FITC fluorescence data with ultrastructural information to map SFTPD localization at the electron microscopy level

  • Photooxidation techniques convert FITC signal to electron-dense deposits visible by electron microscopy

• Expansion microscopy:

  • Physical expansion of specimens preserves FITC fluorescence while achieving effective super-resolution through physical magnification

  • Particularly valuable for mapping SFTPD distribution across larger tissue areas while maintaining subcellular resolution

These advanced imaging approaches are revealing new insights into SFTPD function in normal physiology and disease states, particularly in understanding the protein's role in antimicrobial defense and surfactant homeostasis.

What innovative applications are emerging for SFTPD Antibody, FITC conjugated in translational research?

SFTPD Antibody, FITC conjugated is finding novel applications in translational research areas:

• Immune cell interactions:

  • Tracking SFTPD binding to immune cells using flow cytometry and imaging cytometry

  • Investigating SFTPD-mediated phagocytosis of pathogens by macrophages and neutrophils

  • Studying the role of SFTPD in modulating dendritic cell function and adaptive immunity

• Biomarker development:

  • Quantitative assessment of SFTPD levels in bronchoalveolar lavage fluid as predictive biomarkers for respiratory diseases

  • Flow cytometric quantification of cell-bound SFTPD in patient samples

  • Correlation of SFTPD expression patterns with disease progression and treatment response

• Therapeutic targeting approaches:

  • pH-dependent targeting strategies similar to FITC-pHLIP conjugates that exploit the acidity of disease microenvironments

  • Development of SFTPD-targeted drug delivery systems for lung-specific therapeutics

  • Monitoring biodistribution of SFTPD-targeted therapeutics using in vivo imaging

• Multi-omics integration:

  • Combining FITC-based SFTPD imaging with transcriptomics and proteomics to develop comprehensive models of surfactant biology

  • Single-cell correlation of SFTPD expression with other cellular parameters

  • Integration with mass cytometry (CyTOF) data for high-dimensional analysis of SFTPD-expressing cells

These emerging applications highlight the continuing relevance of FITC-conjugated antibodies in modern research, with SFTPD serving as an important target for understanding pulmonary biology and developing new diagnostic and therapeutic approaches.