WISP3 Antibody, FITC conjugated

What is WISP3 and what biological functions does it serve?

WISP3 (also known as CCN6) belongs to the CCN family of growth factors, which includes connective tissue growth factor (CTGF) and Cyr61. The protein plays essential roles in mitochondrial electron transport and respiration, contributing to normal postnatal skeletal growth and cartilage homeostasis . WISP3 contains four conserved cysteine-rich domains and functions as a dimer in its active form. It is primarily a secreted protein involved in cartilage development and has been implicated in progressive pseudorheumatoid dysplasia, a skeletal disorder affecting cartilage homeostasis by disrupting chondrocyte growth and normal cell columnar organization .

What applications are FITC-conjugated WISP3 antibodies suitable for?

FITC-conjugated WISP3 antibodies are particularly valuable for fluorescence-based techniques. These applications include western blotting (WB, dilution 1:300-5000), immunofluorescence with paraffin-embedded tissue sections (IF/IHC-P, dilution 1:50-200), immunofluorescence with frozen sections (IF/IHC-F, dilution 1:50-200), and immunocytochemistry (IF/ICC, dilution 1:50-200) . The fluorescent conjugation eliminates the need for secondary antibodies in these applications, streamlining experimental workflows and reducing background signal in complex tissue samples.

What is the typical reactivity spectrum of commercially available WISP3 antibodies?

Commercially available WISP3 antibodies show varied reactivity profiles. For example, the FITC-conjugated polyclonal antibody from Bioss (bs-12380R-FITC) has confirmed reactivity with rat samples and predicted reactivity with human, mouse, dog, sheep, horse, rabbit, and monkey samples based on sequence homology . Other WISP3 antibodies, such as Abcam's EPR14769 clone, demonstrate reactivity with human, mouse, and rat samples in Western blot applications . Researchers should verify reactivity for their specific species of interest before designing extensive experiments.

How should FITC-conjugated WISP3 antibodies be stored to maintain optimal activity?

FITC-conjugated WISP3 antibodies require specific storage conditions to preserve fluorophore activity and antibody binding capacity. They should be stored at -20°C and protected from light exposure to prevent photobleaching of the FITC fluorophore . The storage buffer typically contains 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% glycerol to maintain stability. To prevent protein degradation from repeated freeze-thaw cycles, it is recommended to aliquot the antibody into multiple vials upon receipt .

How does epitope selection affect WISP3 antibody performance in different applications?

Epitope selection significantly impacts WISP3 antibody performance across various applications. Antibodies targeting different regions of WISP3 demonstrate distinct binding characteristics and application suitability. For instance, antibodies recognizing the middle region (AA 201-372) of human WISP3 may perform differently than those targeting the C-terminal region . In research requiring detection of specific WISP3 isoforms or post-translationally modified variants, epitope selection becomes particularly critical. Researchers should consider whether they need to detect full-length protein or specific domains when selecting between antibodies targeting different epitopes (middle region versus C-terminal regions) to ensure optimal experimental outcomes.

What are the considerations when using FITC-conjugated WISP3 antibodies in multi-parameter flow cytometry?

When incorporating FITC-conjugated WISP3 antibodies into multi-parameter flow cytometry panels, several methodological considerations are essential. FITC emits in the green spectrum (peak emission ~520 nm), which may overlap with other common fluorophores like PE or GFP . Proper compensation controls are critical to correct for spectral overlap. Additionally, since WISP3 is primarily a secreted protein, permeabilization protocols must be optimized to access intracellular and secretory pathway-associated WISP3. For co-localization studies examining WISP3 alongside mitochondrial markers (given WISP3's role in mitochondrial function), researchers should carefully select fluorophores with minimal spectral overlap and validate antibody performance using appropriate positive and negative controls before conducting comprehensive analyses.

How can researchers validate WISP3 antibody specificity in experimental systems?

Validating WISP3 antibody specificity requires a multi-faceted approach. First, researchers should perform western blots with positive control lysates known to express WISP3 (e.g., Caco-2, HepG2 cell lysates for human samples; mouse brain/heart lysates for murine samples), looking for the expected band size of approximately 39-45 kDa . WISP3 knockout/knockdown models provide essential negative controls to confirm antibody specificity. For FITC-conjugated antibodies specifically, peptide competition assays can determine whether binding is blocked by the immunizing peptide. Additionally, orthogonal detection methods (e.g., mass spectrometry identification of immunoprecipitated proteins) provide further validation. Researchers should be aware that observed band sizes may differ from predicted sizes (39 kDa predicted vs. 45 kDa observed) due to post-translational modifications .

What are the optimal fixation and permeabilization protocols for FITC-conjugated WISP3 antibody immunofluorescence?

Optimal fixation and permeabilization protocols for FITC-conjugated WISP3 antibody immunofluorescence depend on the subcellular localization being investigated. Since WISP3 is a secreted protein that also functions in mitochondrial processes, a balanced approach is necessary . For paraffin-embedded tissues (IHC-P), standard antigen retrieval using citrate buffer (pH 6.0) followed by permeabilization with 0.1-0.3% Triton X-100 is generally effective. For cultured cells (ICC), 4% paraformaldehyde fixation (10-15 minutes at room temperature) followed by gentle permeabilization (0.1% Triton X-100 for 5-10 minutes) helps maintain cellular architecture while allowing antibody access. Extended permeabilization times may be needed when investigating WISP3 in secretory pathway compartments. Researchers should optimize these parameters empirically for their specific cell types and tissues of interest.

How can researchers quantitatively analyze WISP3 expression in tissue samples using FITC-conjugated antibodies?

Quantitative analysis of WISP3 expression using FITC-conjugated antibodies requires standardized image acquisition and analysis protocols. Researchers should establish consistent exposure settings when capturing fluorescence images to enable accurate comparisons between samples. For tissue sections, multiple representative fields (minimum 5-10 per section) should be imaged using identical parameters . Image analysis software (e.g., ImageJ/FIJI) can then be used to quantify parameters such as mean fluorescence intensity, percent positive area, or number of positive cells. For more precise quantification, co-staining with cellular markers can enable cell type-specific WISP3 expression analysis. Flow cytometry provides an alternative quantitative approach for cell suspensions, measuring the median fluorescence intensity of WISP3-FITC staining across different experimental conditions. In all cases, appropriate negative controls and calibration standards should be included to ensure reliable quantification.

What controls should be included when using FITC-conjugated WISP3 antibodies in immunofluorescence studies?

A comprehensive control strategy is essential when using FITC-conjugated WISP3 antibodies in immunofluorescence studies. Primary controls should include: (1) isotype controls (FITC-conjugated rabbit IgG at matching concentrations) to assess non-specific binding; (2) known positive tissue/cells with established WISP3 expression (e.g., chondrocytes for skeletal studies) ; and (3) known negative tissues/cells where possible. Technical controls should include: (1) autofluorescence controls (samples processed without any antibody) to establish background fluorescence levels; (2) peptide competition controls where the antibody is pre-incubated with excess immunizing peptide to confirm binding specificity; and (3) serial dilution controls to determine optimal antibody concentration. For studies examining WISP3's role in progressive pseudorheumatoid dysplasia, including both normal and pathological samples provides important biological reference points for interpreting staining patterns and intensities.

How should researchers address potential cross-reactivity when using WISP3 antibodies in multi-protein studies?

Addressing cross-reactivity concerns in multi-protein studies with WISP3 antibodies requires systematic validation approaches. First, researchers should review the immunogen sequence used to generate the WISP3 antibody (e.g., KLH conjugated synthetic peptide from human WISP3 residues 221-320/354) and conduct bioinformatic analyses to identify proteins with similar epitopes that might cause cross-reactivity. When examining WISP3 alongside other CCN family members (WISP1, WISP2, etc.) that share conserved domains, sequential immunostaining or fluorophore-specific imaging can help distinguish individual signals. For multiplex studies, testing the WISP3 antibody in samples with known expression profiles of related proteins can reveal potential cross-reactivity. Additionally, comparing staining patterns from multiple antibodies targeting different WISP3 epitopes provides confidence that observed signals represent true WISP3 expression rather than cross-reactivity with other proteins.

What are common issues encountered with FITC-conjugated WISP3 antibodies and how can they be resolved?

Several common issues may arise when working with FITC-conjugated WISP3 antibodies. Weak or absent signal often results from suboptimal antibody concentration, inadequate permeabilization (particularly important for detecting secreted WISP3), or epitope masking during fixation . This can be addressed by titrating antibody concentrations (testing ranges from 1:50 to 1:200 for immunofluorescence), optimizing permeabilization protocols, or exploring alternative fixation methods. Photobleaching of the FITC fluorophore is another common issue, resolved by minimizing light exposure during processing, using anti-fade mounting media, and capturing images promptly after staining. High background may result from non-specific binding, which can be mitigated by including blocking steps with 5-10% normal serum from the same species as the secondary antibody would be (if using a detection system) and optimizing washing steps (increasing number and duration of washes) .

Why might observed WISP3 band sizes differ from predicted molecular weights in Western blot analysis?

Discrepancies between observed and predicted WISP3 band sizes in Western blot analysis are commonly reported and can occur for several reasons. The predicted molecular weight of WISP3 is approximately 39 kDa, but observed bands often appear at around 45 kDa . This difference may be attributed to post-translational modifications such as glycosylation, phosphorylation, or other covalent additions that increase apparent molecular weight. Alternative splicing of WISP3 may also generate isoforms with different molecular weights. Additionally, the presence of the four conserved cysteine-rich domains in WISP3 can affect protein migration in SDS-PAGE due to incomplete denaturation of disulfide bonds . When troubleshooting unexpected band sizes, researchers should consider sample preparation conditions (reducing vs. non-reducing), buffer compositions, and pretreatment of samples with glycosidases or phosphatases to determine the source of molecular weight shifts.

How can researchers optimize staining protocols when WISP3 expression levels are low or variable between samples?

Optimizing detection of low or variable WISP3 expression requires systematic protocol refinement. For immunofluorescence applications with FITC-conjugated antibodies, signal amplification strategies can be employed, including: (1) increasing primary antibody concentration (within the recommended range of 1:50-1:200) ; (2) extending incubation times (overnight at 4°C rather than 1-2 hours at room temperature); and (3) using high-sensitivity detection systems. For Western blotting, loading higher protein amounts (up to 30-50 μg per lane versus standard 10-20 μg), extending exposure times, and using enhanced chemiluminescence substrates can improve detection of low-abundance WISP3. When comparing samples with variable WISP3 expression, normalization to appropriate housekeeping proteins or total protein staining is essential for accurate quantification. Additionally, enrichment strategies such as immunoprecipitation prior to Western blotting can concentrate WISP3 from samples with low expression levels, enabling more reliable detection and comparison.

What strategies can address non-specific background when using FITC-conjugated WISP3 antibodies in highly autofluorescent tissues?

Addressing non-specific background and autofluorescence is particularly challenging when using FITC-conjugated antibodies in tissues with high intrinsic autofluorescence (such as brain, kidney, or tissues containing lipofuscin). Several strategies can mitigate these issues: (1) tissue pretreatment with Sudan Black B (0.1-0.3% in 70% ethanol) or copper sulfate (1-10 mM CuSO₄ in 50 mM ammonium acetate) can significantly reduce autofluorescence; (2) spectral imaging and linear unmixing can computationally separate FITC signal from autofluorescence based on their spectral profiles; (3) time-gated detection can exploit the typically longer fluorescence lifetime of FITC compared to autofluorescence; and (4) considering alternative conjugates with fluorescence in different spectral ranges (e.g., far-red) where tissue autofluorescence is less prominent. Including proper autofluorescence controls (unstained sections) is essential for establishing background levels and determining the true specific signal in challenging tissue types.

How should researchers interpret differential WISP3 localization patterns in relation to its diverse biological functions?

Interpreting differential WISP3 localization patterns requires consideration of its multiple reported functions. As a secreted protein involved in both extracellular signaling and mitochondrial function, WISP3 may exhibit complex localization patterns that vary by cell type and physiological state . Co-localization studies with compartment-specific markers can help distinguish between WISP3 populations associated with secretory pathways (using markers like KDEL for ER or TGN46 for trans-Golgi network) versus mitochondrial association (using markers like TOMM20 or MitoTracker dyes). When analyzing tissues affected by progressive pseudorheumatoid dysplasia, researchers should evaluate changes in both extracellular and intracellular WISP3 distribution patterns compared to healthy controls. Quantitative co-localization analysis using tools like JACoP (Just Another Co-localization Plugin) for ImageJ can provide Pearson's correlation coefficients and Manders' overlap coefficients to measure the degree of spatial correlation between WISP3 and various cellular compartments.