SOX8 Antibody, FITC conjugated

What is SOX8 and why is it significant in research?

SOX8 is a transcription factor that plays crucial roles in gene expression regulation during development, particularly in differentiating various cell types. It functions primarily in the nucleus, binding to specific DNA sequences to modulate target gene transcription . SOX8 has been implicated in several important biological processes, including colorectal carcinoma development through FZD6-dependent Wnt/β-catenin signaling , M cell maturation in the intestinal epithelium for immune responses , and ear morphogenesis during embryonic development . As a member of the evolutionarily conserved SOX gene family, SOX8 dysregulation has been linked to various human diseases, highlighting its importance in both normal physiology and pathology .

What are the typical applications for SOX8 antibodies in research?

SOX8 antibodies are commonly used in several research applications:

• Immunofluorescence (IF)/Immunocytochemistry (ICC) for cellular localization studies

• Western blotting (WB) for protein expression analysis

• Immunoprecipitation (IP) for protein interaction studies

• Enzyme-linked immunosorbent assay (ELISA) for quantitative protein detection

• Chromatin immunoprecipitation for studying SOX8 binding to DNA targets, as demonstrated in studies examining SOX8's direct binding to promoter regions of genes like GP2

What is the advantage of using FITC-conjugated SOX8 antibodies compared to unconjugated versions?

FITC-conjugated SOX8 antibodies offer several distinct advantages for researchers:

• They eliminate the need for secondary antibody incubation steps, reducing experiment time and potential for cross-reactivity

• Direct visualization allows for streamlined experimental workflows in immunofluorescence applications

• They enable multi-color imaging when combined with antibodies conjugated to spectrally distinct fluorophores

• The conjugation provides consistent signal intensity by maintaining a defined fluorophore-to-antibody ratio

• They produce cleaner backgrounds in some applications due to reduced non-specific binding associated with secondary antibodies

What are the recommended dilution ranges for FITC-conjugated SOX8 antibodies in immunofluorescence applications?

How should samples be prepared for optimal SOX8 detection in different tissue and cell types?

Sample preparation varies by application and target tissue:

For fixed cell IF/ICC applications:

• Fix cells using 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes (critical for nuclear transcription factors like SOX8)

• Block with 1-5% BSA or normal serum from the same species as the secondary antibody

• For SOX8 specifically, optimize permeabilization as it is primarily located in the nucleus

• For colorectal carcinoma cell lines like HCT116 and SW620 (where SOX8 is upregulated), ensure complete permeabilization to access nuclear proteins

For tissue sections:

• Use either frozen sections (typically 5-10 μm) or formalin-fixed paraffin-embedded (FFPE) sections

• For FFPE sections, perform antigen retrieval (heat-induced epitope retrieval in citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Permeabilize and block as described above

• When examining intestinal tissues for M cell analysis, whole-mount immunofluorescence staining followed by quantitative image cytometry may be optimal as used in studies of Sox8 expression in intestinal M cells

What are the optimal storage conditions for maintaining FITC-conjugated SOX8 antibodies?

To maintain the quality and performance of FITC-conjugated SOX8 antibodies:

• Store at -20°C and avoid freeze-thaw cycles by preparing small aliquots

• Protect from light exposure as fluorophores are light-sensitive and can photobleach

• For longer-term storage, maintain antibodies in storage buffer (typically PBS with 50% Glycerol, preservatives like 0.05% Proclin300, and stabilizers like 0.5% BSA, pH 7.3)

• Aliquoting is generally recommended, though for some formulations it may be unnecessary for -20°C storage

• Working dilutions should be prepared fresh and used within 24 hours when stored at 4°C

How can SOX8 antibodies be used to investigate the role of SOX8 in colorectal carcinoma?

Based on recent research showing SOX8's oncogenic role in colorectal carcinoma:

• Expression analysis: Use FITC-conjugated SOX8 antibodies to compare nuclear SOX8 levels between normal colonic epithelium and CRC tissues using quantitative immunofluorescence

• Co-localization with Wnt pathway components: Perform dual immunofluorescence with SOX8 and β-catenin antibodies to assess nuclear co-localization, supporting the connection between SOX8 and Wnt/β-catenin signaling

• Functional studies: After SOX8 knockdown in CRC cell lines (as demonstrated using specific shRNAs against SOX8), use SOX8 antibodies to confirm knockdown efficiency before phenotypic assays

• Correlation with FZD6 expression: Combine SOX8 and FZD6 immunostaining to investigate their relationship in patient samples, given that SOX8 activates FZD6-dependent Wnt/β-catenin signaling

Researchers should include appropriate controls in these experiments, such as shRNA knockdown validation and correlation with other methodologies like RT-qPCR for mRNA expression.

What are common troubleshooting approaches for weak or absent SOX8-FITC signals?

When encountering weak or absent SOX8-FITC signals:

• Insufficient permeabilization: Increase Triton X-100 concentration or permeabilization time to enhance nuclear access

• Inadequate antigen retrieval: For FFPE samples, optimize antigen retrieval methods (time, temperature, buffer composition)

• Antibody concentration: Try a more concentrated antibody dilution (e.g., 1:50 instead of 1:200)

• Fixation issues: Overfixation can mask epitopes; try reducing fixation time or alternative fixation methods

• Photobleaching: Minimize exposure to light during processing and use anti-fade mounting media

• Low target expression: Confirm SOX8 expression in your sample type; consider positive controls like HepG2 cells which have demonstrated SOX8 reactivity

• Batch variability: Test a new antibody lot or alternative clone if persistent issues occur

How can SOX8-FITC antibodies be integrated into multi-parameter immunofluorescence protocols?

For complex multi-parameter studies:

• Spectral compatibility: FITC/CL488 has excitation/emission maxima around 493/522 nm , making it compatible with other fluorophores like DAPI (nuclei), Cy3/TRITC (red channel), and far-red dyes

• Sequential staining: For multiple primary antibodies from the same species, use sequential staining with intermediate blocking steps

• Multiplexing example protocol:

  • First round: SOX8-FITC antibody (1:100) + rabbit anti-FZD6 + appropriate secondary antibody

  • Counterstain nuclei with DAPI

  • This approach enables simultaneous visualization of SOX8, FZD6 (its downstream target), and nuclear morphology

• Cross-talk prevention: Ensure proper filter sets to prevent bleed-through between channels and use sequential scanning in confocal microscopy

• Signal amplification: For weak signals, consider tyramide signal amplification (TSA) systems compatible with FITC

What are the expected subcellular localization patterns for SOX8 in different cell types?

SOX8 is primarily localized in the nucleus where it functions as a transcription factor . Expected staining patterns include:

• Nuclear localization: Strong, often diffuse nuclear staining is typical, with potential nucleolar exclusion

• Cell type variations:

  • In colorectal carcinoma cells (HCT116, SW620): Prominent nuclear staining with upregulated expression compared to normal colorectal epithelium

  • In M cells within intestinal epithelium: Nuclear localization with expression levels maintained throughout maturation stages

  • In ear development models: Nuclear staining in otic epithelium progenitors

• Expression level gradient: In intestinal M cells, Sox8 expression remains relatively constant or slightly increases along the crypt-dome axis, unlike Spi-B which declines in GP2-high mature M cells

Unexpected cytoplasmic staining might indicate antibody cross-reactivity or issues with experimental conditions.

How can quantitative analysis of SOX8 expression be performed using immunofluorescence data?

For quantitative analysis of SOX8 immunofluorescence:

• Image acquisition standardization:

  • Use identical exposure settings for all samples

  • Include calibration standards if absolute quantification is needed

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

• Nuclear signal quantification methods:

  • Mean fluorescence intensity (MFI) of nuclear SOX8-FITC signal

  • Integrated density (product of area and mean gray value)

  • Nuclear/cytoplasmic signal ratio to control for background

• Analysis workflow:

  • Segment nuclei using DAPI channel

  • Create nuclear masks to measure SOX8-FITC intensity within nuclei

  • Apply threshold to identify positive vs. negative nuclei

  • Quantify percentage of SOX8-positive cells and intensity distributions

• Advanced approaches:

  • Whole-mount immunofluorescence followed by quantitative image cytometry for complex tissues (as used in M cell studies)

  • Single-cell analysis correlating SOX8 expression with spatial information (e.g., distance from crypt base in intestinal epithelium studies)

How do SOX8 expression patterns differ across developmental stages and disease states?

Based on the literature, SOX8 expression varies significantly:

• Developmental contexts:

  • In ear development: Sox8 is among the earliest otic epithelial placode transcription factors, forming a regulatory circuit with Pax2, Lmx1a, and Zbtb16

  • In intestinal M cells: Sox8 is constitutively expressed during M cell development, from early differentiation stages through maturation

• Disease states:

  • In colorectal carcinoma: SOX8 expression is upregulated in CRC cell lines and tumor tissues compared to normal tissues

  • Knockdown of SOX8 in CRC reduces cell proliferation, migration, and invasion capabilities

• Functional impact:

  • In Sox8-deficient mice: Marked decrease in mature M cells, resulting in reduced antigen uptake in Peyer's patches and attenuated germinal center reactions

  • In CRC models: SOX8 activates Wnt/β-catenin signaling through FZD6 upregulation

When analyzing SOX8 expression patterns, researchers should consider both the intensity of staining and the percentage of positive cells, as both parameters may change independently in different biological contexts.

How can FITC-conjugated SOX8 antibodies be used to investigate SOX8's role in transcriptional regulation?

FITC-conjugated SOX8 antibodies can be powerful tools for studying SOX8's transcriptional functions:

• Chromatin dynamics studies:

  • Combined immunofluorescence for SOX8-FITC with other fluorescently-labeled transcription factors or chromatin modifiers

  • Analysis of nuclear distribution patterns during cell cycle progression or differentiation

• Transcriptional complex visualization:

  • Proximity ligation assay (PLA) using SOX8-FITC antibodies with antibodies against suspected binding partners

  • This approach can visualize protein-protein interactions within 40nm distance in situ

• Combined approaches:

  • Correlative light and electron microscopy to relate SOX8 distribution to nuclear ultrastructure

  • Live cell imaging with transiently transfected fluorescent protein-tagged SOX8 followed by fixation and immunostaining with SOX8-FITC antibodies to validate expression patterns

• Transcriptional target validation:

  • Immunofluorescence to correlate SOX8 expression with target gene products, such as FZD6 in colorectal cancer cells or GP2 in M cells

What experimental designs can validate SOX8 antibody specificity for critical research applications?

Thorough validation of SOX8 antibody specificity is crucial:

• Genetic approaches:

  • Test antibody staining in Sox8-knockout tissues/cells (as demonstrated for specificity validation in Sox8-deficient mice)

  • Use siRNA/shRNA-mediated SOX8 knockdown to confirm signal reduction with multiple shRNAs targeting different regions

• Peptide competition assays:

  • Pre-incubate SOX8 antibody with immunizing peptide before application to samples

  • Signal should be significantly reduced if antibody is specific

• Comparison across antibody clones:

  • Compare staining patterns using multiple antibodies targeting different SOX8 epitopes

  • Consistent patterns across antibodies support specificity

• Cross-species validation:

  • Test antibody in species with high SOX8 sequence homology

  • Correlate with evolutionary conservation of recognition epitope

• Orthogonal validation:

  • Correlate protein detection with mRNA expression (ISH/qPCR)

  • Combine with functional assays demonstrating biological relevance of detected protein