DICER1 Antibody, FITC conjugated

What is DICER1 and why is it significant in research applications?

DICER1 is a double-stranded RNA (dsRNA) endoribonuclease that plays a central role in short dsRNA-mediated post-transcriptional gene silencing. It cleaves naturally occurring long dsRNAs and short hairpin pre-microRNAs (miRNA) into fragments of twenty-one to twenty-three nucleotides with 3' overhang of two nucleotides, producing short interfering RNAs (siRNA) and mature microRNAs respectively. These siRNAs and miRNAs serve as guides to direct the RNA-induced silencing complex (RISC) to complementary RNAs for degradation or translation prevention. This process is crucial in controlling mobile and repetitive DNA elements in the genome and in degrading exogenous RNA of viral origin. The miRNA pathway specifically regulates target gene expression, making DICER1 a critical research target for understanding fundamental RNA processing mechanisms .

What applications is a FITC-conjugated DICER1 antibody suitable for?

FITC-conjugated DICER1 antibodies are particularly valuable for applications requiring direct fluorescence detection without secondary antibodies. Based on available validation data, these antibodies are suitable for:

• Immunofluorescence and Immunocytochemistry (IF/ICC): Particularly effective for subcellular localization studies with recommended dilutions of 1:50-200 for IF(IHC-P)

• Flow Cytometry (FC): Allowing direct detection of DICER1 in fixed and permeabilized cells

• Fluorescence microscopy: Enabling direct visualization of DICER1 expression patterns

While the FITC-conjugated variant has these specific applications, related non-conjugated DICER1 antibodies have been validated for additional techniques including Western Blot (1:200-1:1000), Immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg protein), and ELISA (0.1-0.5 μg/ml) .

What cell lines and tissues have been validated for DICER1 antibody applications?

Multiple cell lines and tissues have been validated for various DICER1 antibody applications:

The FITC-conjugated antibody (BS-6697R-FITC) shows reactivity with human, mouse, and rat samples per manufacturer specifications .

What are the storage conditions and stability considerations for DICER1 antibodies?

DICER1 antibodies require specific storage conditions to maintain reactivity and performance. The recommended storage is at -20°C in buffer containing PBS with 0.02% sodium azide and 50% glycerol pH 7.3. Under these conditions, antibodies remain stable for one year after receipt. For FITC-conjugated antibodies, additional precautions should be taken to protect from light exposure, as fluorophores are susceptible to photobleaching. Repeated freeze-thaw cycles should be avoided as they can degrade antibody quality and reduce binding efficiency. For working solutions, storage at 4°C for short periods (1-2 weeks) is acceptable, but for longer-term storage, aliquoting is recommended to minimize freeze-thaw cycles .

How can researchers validate the specificity of DICER1 antibodies in experimental designs?

Validating DICER1 antibody specificity requires a multi-faceted approach:

• Positive and negative control samples: Use cell lines with known DICER1 expression (e.g., K562, HepG2, MCF-7) as positive controls. For negative controls, employ DICER1 knockdown or knockout samples.

• Multiple detection methods: Cross-validate findings using at least two independent techniques (e.g., Western blot plus immunofluorescence).

• Band size verification: For Western blot applications, confirm detection at the appropriate molecular weight (219 kDa calculated, with observed bands at 220-250 kDa and sometimes at 90 kDa, potentially representing cleavage products) .

• Antibody validation controls:

  • Primary antibody omission control

  • Isotype control antibody (e.g., rabbit IgG at matching concentration)

  • Blocking peptide competition assay

• Orthogonal validation: Compare protein expression with mRNA expression data or proteomics data.

For FITC-conjugated antibodies specifically, additional controls include unstained samples and samples stained with isotype-matched FITC-conjugated control antibodies to establish autofluorescence baselines and non-specific binding .

What are the key considerations when studying DICER1 mutations in cancer research?

Research into DICER1 mutations, particularly in sarcomas and other cancers, requires careful experimental design considerations:

• Mutation patterns: DICER1 frequently exhibits biallelic mutations in tumors, with one allele often containing a germline (or somatic) truncating mutation and the second allele harboring a somatic missense mutation in the RNase IIIa or RNase IIIb domain. These hotspot mutations (particularly in positions such as G1809R, D1709N, D1713V, and E1813G) affect metal ion binding and catalytic activity .

• Sequencing approach: Complete gene sequencing is recommended rather than hotspot screening, as mutations can occur throughout the coding region. The RNase III domains are particularly important to examine.

• Sample considerations: Both tumor and matched normal tissue should be sequenced to distinguish somatic from germline mutations. In the study of sarcomas, researchers identified cases where mutations occurred in trans (on different alleles) leading to complete loss of functional DICER1 .

• Clinical correlation: DICER1 mutations may be associated with DICER1 syndrome, a cancer predisposition condition. Research should document additional clinical findings such as cystic nephroma (CN), multinodular goiter (MNG), and pleuropulmonary blastoma (PPB) .

The table below summarizes findings from sarcoma research that identified DICER1 mutations:

These patterns suggest specific mechanisms of DICER1 dysfunction in tumorigenesis that are important to consider in experimental designs .

How do FITC-conjugated DICER1 antibodies compare with other detection methods for studying DICER1 in cellular contexts?

FITC-conjugated DICER1 antibodies offer distinct advantages and limitations compared to other detection methods:

Advantages:

• Direct detection: No secondary antibody is required, reducing protocol steps and potential cross-reactivity

• Multiplexing capability: FITC's emission spectrum (peak ~520nm) allows combination with other fluorophores in multi-color experiments

• Established detection systems: Most fluorescence microscopes and flow cytometers have standard FITC filter sets

Limitations:

• Signal amplification: Direct conjugates typically provide lower signal amplification compared to systems using secondary antibodies

• Photobleaching: FITC is more susceptible to photobleaching than some alternative fluorophores

• Fixed sensitivity: Unlike enzyme-based detection methods (e.g., HRP), signal amplification cannot be increased through longer substrate development

For optimal detection in different research contexts, consider:

• For high sensitivity requirements in low-expression systems: HRP-conjugated detection systems may be preferable

• For co-localization studies: FITC-conjugated antibodies combined with spectrally distinct fluorophores (e.g., Cy3, Cy5) are ideal

• For flow cytometry applications: FITC-conjugated antibodies work well but require careful compensation when multiplexing

What protocol optimizations are recommended for using FITC-conjugated DICER1 antibodies in flow cytometry?

For optimal results with FITC-conjugated DICER1 antibodies in flow cytometry, consider these methodological refinements:

• Cell fixation and permeabilization: As DICER1 is primarily intracellular, effective permeabilization is crucial. Use 4% paraformaldehyde for fixation followed by a permeabilization buffer for intracellular staining, as demonstrated in the Caco-2 cell protocol .

• Antibody concentration: Start with the recommended concentration of 1-3 μg per 1×10^6 cells, but titrate to optimize signal-to-noise ratio for your specific cell type.

• Blocking: Include a blocking step with 10% normal goat serum (or serum matching the species of secondary antibody if used) to reduce non-specific binding.

• Controls: Include these essential controls:

  • Unstained cells (for autofluorescence baseline)

  • Isotype control (rabbit IgG at 1 μg/1×10^6 cells)

  • Single-color controls (for compensation when multiplexing)

• Incubation conditions: Optimal incubation is 30 minutes at 20°C; longer incubations or higher temperatures may increase non-specific binding.

• Washing steps: Include at least three thorough washing steps after antibody incubation to reduce background.

• Instrument settings: Adjust the PMT voltage for FITC detection to place the negative population in the first decade of the logarithmic scale.

When analyzing data, use appropriate gating strategies to exclude debris and doublets before examining DICER1 expression patterns .

What are the recommended sample preparation methods for immunofluorescence using DICER1 antibodies?

For optimal immunofluorescence detection of DICER1, sample preparation should follow these methodological guidelines:

• Fixation options:

  • For cultured cells: 4% paraformaldehyde for 15-20 minutes at room temperature

  • For tissue sections: 10% neutral buffered formalin followed by paraffin embedding

• Antigen retrieval for tissue sections: Two options have been validated:

  • TE buffer pH 9.0 (recommended primary option)

  • Citrate buffer pH 6.0 (alternative method)

  • For enzyme-based retrieval: Follow IHC enzyme antigen retrieval protocol for 15 minutes

• Permeabilization:

  • For cultured cells: 0.1-0.5% Triton X-100 in PBS for 5-10 minutes

  • For tissue sections: Permeabilization is often accomplished during antigen retrieval

• Blocking: 10% goat serum (or serum from the species of the secondary antibody) for 30-60 minutes at room temperature.

• Antibody incubation:

  • Primary antibody: For non-conjugated antibodies, use 1:50-1:500 dilution for IF-P or 1:200-1:800 for IF/ICC; for FITC-conjugated antibodies, use 1:50-200

  • For non-conjugated primary antibodies, follow with appropriate fluorophore-conjugated secondary antibody (e.g., DyLight®488 Conjugated Goat Anti-Rabbit IgG at 1:100 dilution)

  • Incubate primary antibody overnight at 4°C for best results

• Nuclear counterstaining: DAPI is recommended for nuclear visualization, which provides contrast to cytoplasmic DICER1 staining.

• Mounting: Use anti-fade mounting medium specifically formulated for fluorescence microscopy to minimize photobleaching of the FITC signal .

How can researchers troubleshoot non-specific binding or weak signals when using DICER1 antibodies?

When encountering issues with DICER1 antibody performance, systematic troubleshooting approaches can resolve both non-specific binding and weak signal problems:

For Non-Specific Binding:

• Increase blocking stringency:

  • Extend blocking time to 1-2 hours

  • Increase blocking serum concentration to 10-15%

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

• Optimize antibody dilution:

  • Further dilute primary antibody (try 2-5× more dilute than recommended)

  • Consider shorter incubation times at room temperature instead of overnight at 4°C

• Enhance washing protocols:

  • Increase number of washes (5-6 washes)

  • Use 0.05-0.1% Tween-20 in wash buffer

  • Extend wash durations to 10 minutes per wash

• Pre-absorption:

  • Pre-absorb the antibody with cell/tissue lysate from a negative control sample

For Weak Signals:

• Antigen retrieval optimization:

  • Try both recommended methods: TE buffer pH 9.0 and citrate buffer pH 6.0

  • Extend retrieval time (15-20 minutes)

  • Ensure proper temperature maintenance during retrieval

• Antibody concentration:

  • Use the upper end of the recommended concentration range (e.g., 1:50 for IHC)

  • For Western blot, increase to 1:200 dilution from the recommended 1:200-1:1000 range

• Signal enhancement:

  • For immunofluorescence: Use a more sensitive detection system like tyramide signal amplification

  • For colorimetric detection: Extend substrate development time

• Sample handling:

  • Minimize time between sample collection and fixation

  • Ensure proper fixation times (over-fixation can mask epitopes)

  • Store FFPE blocks properly to prevent antigen degradation

• Fluorophore considerations (for FITC-conjugated antibodies):

  • Protect from light during all steps

  • Use fresh antibody aliquots to avoid repeated freeze-thaw cycles

  • Adjust microscope settings to optimize detection sensitivity

What controls should be included when designing experiments with DICER1 antibodies?

Robust experimental design with DICER1 antibodies requires comprehensive controls to ensure data reliability and interpretability:

Essential Controls for All Applications:

• Positive controls:

  • Cell lines with confirmed DICER1 expression: HeLa, K-562, HepG2, MCF-7

  • Tissue types with validated expression: human testis, human ovary tumor tissue

• Negative controls:

  • Primary antibody omission: Samples processed identically but with antibody diluent only

  • Isotype control: Irrelevant rabbit IgG at the same concentration as DICER1 antibody

  • DICER1 knockdown/knockout controls (if available): Samples with experimentally reduced DICER1 expression

Application-Specific Controls:

• For Western Blot:

  • Loading control: Antibody against housekeeping protein (β-actin, GAPDH)

  • Molecular weight marker: To confirm correct band size (expected 219 kDa calculated, 220-250 kDa observed)

• For Immunofluorescence/ICC:

  • Counterstain: DAPI for nuclear visualization

  • Autofluorescence control: Sample without any antibody treatment

  • Secondary antibody only control (for non-conjugated primary antibodies)

• For Flow Cytometry:

  • Unstained cells

  • Single-color controls for compensation

  • Fluorescence minus one (FMO) controls

  • Viability dye to exclude dead cells that may bind antibodies non-specifically

• For Immunoprecipitation:

  • Input sample (pre-IP lysate)

  • IgG control IP (using non-specific IgG)

  • Reciprocal IP (if studying protein-protein interactions)

• For DICER1 Mutation Studies:

  • Matched normal tissue for comparison to tumor samples

  • Known mutation-positive control samples if available

How are DICER1 antibodies used in cancer research, particularly in the context of DICER1 syndrome?

DICER1 antibodies serve as crucial tools in cancer research, particularly for investigating DICER1 syndrome, a tumor predisposition disorder characterized by germline DICER1 mutations. These antibodies enable several research applications:

• Protein expression analysis: DICER1 antibodies allow researchers to assess protein expression levels and localization in various tumor types. This is particularly important in DICER1 syndrome, where one allele typically contains a germline truncating mutation and the second allele often harbors a somatic missense mutation in the RNase IIIa or RNase IIIb domain .

• Mutation-specific expression patterns: Using immunohistochemistry and immunofluorescence with DICER1 antibodies, researchers can examine how specific mutations affect protein expression patterns. In sarcomas, DICER1 hotspot mutations (such as G1809R, D1709N, D1713V, and E1813G) have been identified, and antibody-based detection can reveal their impact on protein expression and localization .

• Diagnostic and prognostic marker development: DICER1 syndrome is associated with various tumor types including pleuropulmonary blastoma, cystic nephroma, and embryonal rhabdomyosarcoma. DICER1 antibodies help characterize these tumors and may contribute to developing diagnostic or prognostic markers.

• Therapeutic target evaluation: As understanding of DICER1's role in cancer development grows, antibodies enable assessment of potential therapeutic strategies targeting the microRNA processing pathway.

A recent study sequenced DICER1 in 67 sarcomas and identified biallelic somatic mutations in several cases, including embryonal rhabdomyosarcoma (ERMS). These findings highlight the importance of DICER1 antibodies in characterizing the molecular pathology of these rare cancers .

How can DICER1 antibodies be utilized to study microRNA processing pathways in research models?

DICER1 antibodies provide powerful tools for investigating microRNA (miRNA) processing pathways in various research models:

• Co-immunoprecipitation studies: DICER1 antibodies can be used to isolate DICER1 protein complexes, enabling identification of interaction partners in the miRNA processing machinery. Recommended protocols involve using 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate, followed by mass spectrometry or Western blot analysis of co-precipitated proteins .

• Subcellular localization: FITC-conjugated DICER1 antibodies permit direct visualization of DICER1 localization during miRNA processing. This approach reveals how DICER1 distribution changes in response to experimental conditions or disease states. Recommended dilutions for immunofluorescence range from 1:50-1:500 for IF-P or 1:200-1:800 for IF/ICC .

• Expression correlation studies: By combining DICER1 antibody staining with quantification of miRNA levels, researchers can establish correlations between DICER1 protein expression and miRNA abundance in various cellular contexts.

• Functional studies: Following experimental manipulation of DICER1 (knockdown, overexpression, or mutation), antibodies allow confirmation of altered protein levels and subsequent analysis of effects on miRNA processing. Western blot analysis using 1:200-1:1000 dilution can quantify expression changes .

• Cross-species comparative studies: DICER1 antibodies with reactivity to human, mouse, and rat samples (such as BS-6697R-FITC) enable comparative studies across species, providing evolutionary insights into miRNA processing mechanisms .

• Flow cytometry applications: For high-throughput analysis of DICER1 expression in heterogeneous cell populations, flow cytometry using FITC-conjugated antibodies (0.40 μg per 10^6 cells) allows quantitative assessment of expression levels at the single-cell level .

These approaches collectively enhance understanding of DICER1's role in miRNA biogenesis and how disruptions in this pathway contribute to disease pathogenesis.

What methodological approaches can researchers use to study the effects of DICER1 mutations on protein function?

Investigating the functional consequences of DICER1 mutations requires integrated methodological approaches:

• Protein expression and localization analysis:

  • Western blot analysis using DICER1 antibodies (1:200-1:1000 dilution) to assess expression levels and detect truncated proteins

  • Immunofluorescence microscopy with FITC-conjugated antibodies (1:50-200 dilution) to evaluate subcellular localization changes

  • Flow cytometry for quantitative single-cell analysis of expression levels

• Enzymatic activity assays:

  • In vitro processing assays using recombinant wild-type and mutant DICER1 proteins

  • Substrate processing analysis using labeled pre-miRNAs or dsRNAs

  • Quantification of cleavage products through gel electrophoresis or high-throughput sequencing

• Structure-function correlation:

  • Computational modeling of RNase III domains to predict effects of mutations (particularly hotspot mutations like G1809R, D1709N, D1713V, and E1813G)

  • Analysis of metal ion binding and catalytic site integrity in mutant proteins

• miRNA profiling:

  • Small RNA sequencing to analyze global miRNA expression patterns in cells with DICER1 mutations

  • Quantitative PCR to validate changes in specific miRNAs

  • Correlation of miRNA changes with DICER1 protein expression detected by antibodies

• Protein-protein interaction studies:

  • Immunoprecipitation using DICER1 antibodies (0.5-4.0 μg for 1.0-3.0 mg protein) followed by mass spectrometry

  • Yeast two-hybrid or mammalian two-hybrid assays to assess interaction with known partners

  • Proximity ligation assays to visualize interactions in situ

• Functional rescue experiments:

  • Introduction of wild-type DICER1 into mutant backgrounds

  • Creation of domain-specific mutations to isolate functional effects

  • Assessment of miRNA processing restoration through antibody-based detection methods

• In vivo model systems:

  • Generation of mouse models with specific DICER1 mutations found in human cancers

  • Tissue-specific expression analysis using DICER1 antibodies

  • Correlation of phenotypes with molecular changes in miRNA processing

These methodological approaches provide complementary insights into how specific DICER1 mutations affect protein function, helping researchers understand the molecular mechanisms underlying DICER1 syndrome and related disorders.

What are the comparative advantages of using FITC-conjugated versus unconjugated DICER1 antibodies in multiplexed imaging experiments?

When designing multiplexed imaging experiments, researchers should consider these comparative advantages of FITC-conjugated versus unconjugated DICER1 antibodies:

FITC-Conjugated DICER1 Antibodies:

Advantages:

• Direct detection: Eliminates need for secondary antibody, reducing protocol complexity and potential cross-reactivity issues

• Consistent signal: Provides fixed fluorophore:antibody ratio, enhancing quantitative reliability

• Reduced background: Minimizes non-specific binding associated with secondary antibodies

• Simplified multiplexing: Enables easier combination with other antibodies of different host species without cross-reactivity concerns

• Shortened protocol: Eliminates secondary antibody incubation and washing steps, reducing experiment time by approximately 2 hours

Limitations:

• Fixed signal intensity: Cannot be amplified through secondary antibody strategies

• Photobleaching susceptibility: FITC is more prone to photobleaching than some alternative fluorophores

• Limited spectral options: Restricted to FITC's emission spectrum (peak ~520nm)

• Reduced sensitivity: Generally provides lower detection sensitivity than amplified systems

Unconjugated DICER1 Antibodies:

Advantages:

• Signal amplification: Secondary antibodies can provide signal enhancement (typically 2-5× stronger signal)

• Flexibility in detection: Compatible with various secondary antibody conjugates (fluorescent, enzymatic, etc.)

• Adaptable to various applications: Single primary antibody stock can be used across multiple detection methods

• Enhanced sensitivity: Better detection of low-abundance targets through amplification strategies

• Alternative to fluorescence: Can be used with enzymatic detection methods (HRP, AP) not subject to photobleaching

Limitations:

• Increased background risk: Additional antibody layer can introduce non-specific binding

• Cross-reactivity concerns: May limit multiplexing options with antibodies from the same host species

• Longer protocols: Requires additional incubation and washing steps

• Batch variability: Secondary antibody quality can introduce another variable

For optimal multiplexed imaging specifically, FITC-conjugated DICER1 antibodies (BS-6697R-FITC) work best when combined with directly conjugated antibodies of spectrally distinct fluorophores (e.g., Cy3, Cy5, APC) targeting other proteins of interest .

How should researchers approach quantitative analysis of DICER1 expression using immunofluorescence or flow cytometry?

Quantitative analysis of DICER1 expression requires rigorous methodology to ensure reliable and reproducible results:

For Immunofluorescence Analysis:

• Image acquisition standardization:

  • Use identical exposure settings across all samples and conditions

  • Capture multiple fields per sample (minimum 5-10 random fields)

  • Include representative areas while avoiding edge artifacts

  • Apply flat-field correction to account for uneven illumination

• Quantification approaches:

  • Mean fluorescence intensity (MFI) measurement within defined regions of interest

  • Integrated density calculation (area × mean intensity)

  • Cell-by-cell analysis with nuclear counterstain as reference

  • Subcellular compartment analysis (nuclear vs. cytoplasmic distribution)

• Controls for normalization:

  • Include calibration standards in each experiment

  • Use reference cell lines with known DICER1 expression levels

  • Normalize to cellular landmarks (e.g., DAPI for nuclear staining)

• Software tools:

  • ImageJ/FIJI with appropriate plugins for automated analysis

  • CellProfiler for high-throughput cell-based quantification

  • Specialized fluorescence analysis software with colocalization capabilities

For Flow Cytometry Analysis:

• Sample preparation optimization:

  • Standardize fixation and permeabilization protocols

  • Use recommended antibody concentration (0.40 μg per 10^6 cells in 100 μl)

  • Include cell viability dye to exclude dead cells

• Instrument setup:

  • Perform daily quality control using calibration beads

  • Optimize PMT voltages using positive and negative controls

  • Apply compensation when multiplexing with other fluorophores

• Gating strategy:

  • Apply consistent gating across samples

  • Use forward/side scatter to exclude debris and select viable cells

  • Apply singlet gating to exclude doublets

  • Use isotype controls to set positive/negative boundaries

• Data reporting standards:

  • Report median fluorescence intensity (MFI) rather than mean (less sensitive to outliers)

  • Calculate fold change relative to control samples

  • Include both percentage of positive cells and MFI values

• Statistical considerations:

  • Perform at least three independent biological replicates

  • Apply appropriate statistical tests (t-test, ANOVA with post-hoc tests)

  • Report effect sizes alongside p-values