DPPA3 Antibody, FITC conjugated

Basic Research Questions

• What is DPPA3 and why is it important to detect with fluorescent antibodies?

  DPPA3 (Stella/PGC7) is a maternal factor expressed in naïve embryonic stem cells that plays crucial roles in regulating DNA methylation, promoting the 2-cell embryonic-like (2CLC) state, and participating in DNA damage response pathways. FITC-conjugated antibodies enable direct visualization of DPPA3 in immunofluorescence experiments without requiring secondary antibodies. This is particularly valuable when studying DPPA3's subcellular localization, as it closely co-localizes with γH2AX in DNA damage responses and shows enriched expression in specific cell populations like Zscan4-positive cells .

• What are the optimal fixation and permeabilization methods for DPPA3 immunofluorescence?

  For optimal DPPA3 detection, fix cells with 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.2% Triton X-100 for 10 minutes. This method preserves both protein epitopes and cellular morphology. After fixation and permeabilization, wash cells three times with PBS before antibody incubation. For detecting DPPA3 in embryonic stem cells specifically, this protocol allows visualization of its nuclear localization pattern which is critical for understanding its function in chromatin regulation .

• What dilution ratios are recommended for DPPA3-FITC antibodies in different applications?

  For immunofluorescence microscopy, FITC-conjugated DPPA3 antibodies typically work optimally at 1:100 to 1:200 dilution in blocking buffer (3% BSA in PBS). For flow cytometry applications, a 1:200 dilution is generally suitable, as noted in experimental protocols . When performing Western blot analyses, a more concentrated 1:50 to 1:100 dilution may be required. Always titrate the antibody with your specific sample type to determine optimal concentration, as signal intensity can vary between different cell types and experimental conditions.

• How can I distinguish specific DPPA3 signal from background fluorescence?

  To distinguish specific DPPA3 signal:

  • Always include appropriate negative controls (isotype control antibodies conjugated to FITC)

  • Use wild-type cells alongside DPPA3 knockdown cells as biological controls

  • Apply proper blocking (3-5% BSA or normal serum) for at least 1 hour before antibody incubation

  • Include DAPI nuclear counterstain to confirm nuclear localization of DPPA3

  • Consider spectral unmixing if autofluorescence is problematic in your samples

  In embryonic stem cells, authentic DPPA3 signal shows nuclear localization with higher intensity in 2CLC populations and co-localization with DNA damage markers like γH2AX in certain contexts .

• How should samples be stored after staining with DPPA3-FITC antibodies?

  After staining, mount samples in anti-fade mounting medium containing DAPI for nuclear counterstaining. For short-term storage (1-2 weeks), keep slides at 4°C protected from light. For long-term storage, -20°C is recommended, but be aware that even with anti-fade reagents, FITC signal may gradually decrease over time. When quantitative measurements are needed, image all experimental conditions within the same timeframe. Avoid repeated freeze-thaw cycles which can compromise both tissue integrity and fluorescent signal.

Advanced Research Questions

• How can DPPA3-FITC antibodies be used to track the 2C-like state in embryonic stem cell cultures?

  DPPA3-FITC antibodies can be used to identify and isolate 2C-like cells (2CLCs) through:

  Flow cytometry protocol:

  1. Harvest ESCs using accutase to maintain surface epitopes

  2. Fix cells with 2% paraformaldehyde for 10 minutes

  3. Permeabilize with 0.2% Triton X-100 for 10 minutes

  4. Block with 3% BSA for 30 minutes

  5. Incubate with DPPA3-FITC (1:200) and Zscan4 antibody (1:100) for dual staining

  6. Sort cells based on DPPA3-high/Zscan4-positive phenotype

  This approach enables enrichment of the rare 2CLC population (typically 1-5% of ESCs). Research has shown that DPPA3 overexpression significantly increases the percentage of Zscan4-positive cells from approximately 1.12% to higher levels, while DPPA3 knockdown reduces this population to around 0.209% .

• What controls are necessary when studying DPPA3's role in DNA demethylation using fluorescent microscopy?

  When investigating DPPA3's role in DNA demethylation, implement these critical controls:

  Essential controls:

  • Positive control: Cells known to express high DPPA3 (e.g., naïve ESCs)

  • Negative control: DPPA3 knockdown cells (verified ~30% expression level)

  • Methylation status control: Parallel 5mC antibody staining

  • Technical control: Secondary-only and isotype controls

  Experimental design considerations:

  1. Perform quantitative analysis of 5mC foci number per cell alongside DPPA3 staining

  2. Include UHRF1 co-staining to confirm mechanism of action

  3. Consider time-course experiments to track demethylation dynamics

  Research demonstrates that DPPA3 overexpression significantly reduces 5mC foci to less than half compared to control ESCs, confirming its role in promoting DNA hypomethylation .

• How can DPPA3-FITC antibodies be combined with other markers in multi-parameter flow cytometry?

  For multi-parameter flow cytometry with DPPA3-FITC:

  Recommended panel design:

  Marker	Fluorophore	Purpose	Dilution
  DPPA3	FITC	Identify DPPA3+ cells	1:200
  Zscan4	PE	2CLC marker	1:100
  MERVL	APC	2CLC-associated retrotransposon	1:150
  γH2AX	PE-Cy7	DNA damage marker	1:100
  DAPI	Pacific Blue	Live/dead discrimination	1:1000

  Protocol optimization:

  1. Perform single-color controls for compensation

  2. Use Fluorescence Minus One (FMO) controls for gating

  3. Adjust PMT voltages for balanced signal distribution

  4. Analyze correlations between DPPA3 intensity and other markers

  This approach revealed that ESCs with higher DPPA3 expression levels show increased co-expression of Zscan4, confirming the relationship between DPPA3 and the 2C-like state .

• What are the key considerations when using DPPA3 antibodies to investigate chromatin accessibility changes?

  When investigating DPPA3-mediated chromatin changes:

  Integrated protocol approach:

  1. Perform immunofluorescence with DPPA3-FITC antibodies

  2. Process parallel samples for ATAC-Seq analysis

  3. Compare DPPA3 localization with regions of differential accessibility

  Critical analytical considerations:

  • Focus on heterochromatic regions, particularly near Zscan4 cluster and MERVL elements

  • Analyze accessibility at repeat regions (LINE-1, IAP elements)

  • Compare DPPA3 overexpression vs. knockdown effects on chromatin state

  Research demonstrates that DPPA3 overexpression leads to extensive chromatin opening specifically at 2CLC-related gene sets, with ATAC-Seq showing enriched signals at Zscan4 clusters and MERVL elements, which are typically located in heterochromatic regions .

• How can I perform quantitative analysis of DPPA3 expression levels in relation to DNA methylation status?

  For quantitative correlation analysis between DPPA3 and methylation:

  Sequential staining protocol:

  1. First perform DPPA3-FITC immunostaining

  2. Image and record cell positions

  3. Perform 5mC antibody staining on the same sample

  4. Re-image the same fields

  5. Quantify signal intensities using ImageJ/Fiji

  Quantitative analysis approach:

  • Measure nuclear DPPA3-FITC intensity

  • Quantify 5mC foci number and intensity

  • Perform correlation analysis between measurements

  • Bin cells into DPPA3-high, medium, and low expression groups

  This approach confirms the inverse relationship between DPPA3 expression and DNA methylation levels. When quantified, cells with high DPPA3 expression show approximately 50% reduction in 5mC foci compared to controls .

• What experimental approaches can verify DPPA3's direct binding to UHRF1 using fluorescently-labeled antibodies?

  To verify DPPA3-UHRF1 interactions:

  Proximity Ligation Assay (PLA) protocol:

  1. Fix and permeabilize cells as standard

  2. Incubate with DPPA3-FITC antibody (1:150) and anti-UHRF1 antibody (1:100)

  3. Apply PLA probes and perform ligation/amplification

  4. Quantify PLA signal dots per nucleus

  FRET-based approach:

  1. Use DPPA3-FITC as donor fluorophore

  2. Use UHRF1 antibody with compatible acceptor fluorophore

  3. Measure FRET efficiency in different cellular compartments

  These approaches can confirm the direct interaction suggested by research showing DPPA3 binds and displaces UHRF1 from chromatin, thereby preventing maintenance DNA methylation .

• How does DPPA3 antibody staining pattern change during induced pluripotent stem cell (iPSC) reprogramming?

  During iPSC reprogramming, DPPA3 expression follows a specific pattern:

  Temporal dynamics:

  Reprogramming Stage	DPPA3 Expression	Co-markers	Function
  Fibroblast state	Absent	-	-
  Early reprogramming	Low/sporadic	OCT4+/NANOG-	Initial epigenetic changes
  Mid reprogramming	Increasing	OCT4+/NANOG+	DNA demethylation initiation
  Late reprogramming	High, nuclear	OCT4+/NANOG+/Zscan4+	Global hypomethylation
  Naïve state	Highest	2CLC markers+	Maintains hypomethylated state

  Tracking DPPA3 expression via FITC-conjugated antibodies during reprogramming provides a valuable marker for monitoring progression toward the naïve pluripotent state, particularly as cells approach the 2C-like state characterized by global DNA hypomethylation .