CDH1 Antibody, FITC conjugated

What is CDH1 and why is it significant in cancer research?

CDH1 encodes E-cadherin, a calcium-dependent cell adhesion protein essential for maintaining epithelial integrity. Germline CDH1 mutations confer high lifetime risks of developing diffuse gastric cancer (DGC) and lobular breast cancer (LBC). The cumulative risk by age 80 is approximately 70% for men and 56% for women for DGC, and 42% for women for LBC . CDH1 is considered a tumor suppressor gene, and its inactivation through genetic or epigenetic mechanisms is associated with cancer progression, particularly in cancers with the diffuse/infiltrative growth pattern .

What are the optimal fixation conditions for immunofluorescence detection of E-cadherin using FITC-conjugated CDH1 antibodies?

For optimal immunofluorescence detection of E-cadherin using FITC-conjugated antibodies, cells should be fixed with 4% paraformaldehyde for 15-20 minutes at room temperature. Over-fixation can mask epitopes, while under-fixation may compromise cellular morphology. For tissue sections, 10% neutral buffered formalin is recommended with fixation times optimized based on tissue thickness. Permeabilization with 0.1-0.5% Triton X-100 for 5-10 minutes facilitates antibody access to membrane-associated E-cadherin without disrupting epitope structure.

How can I distinguish between membranous and cytoplasmic E-cadherin staining patterns?

Distinguishing between membranous and cytoplasmic E-cadherin staining is crucial as it reflects protein functionality. Normal epithelial tissues show strong, continuous membranous staining at cell-cell junctions. In contrast, many cancers, particularly those with CDH1 mutations, display aberrant cytoplasmic localization or complete loss of expression . For accurate assessment:

• Use confocal microscopy with z-stack imaging to precisely localize the signal

• Include co-staining with membrane markers (e.g., Na+/K+ ATPase) or nuclear counterstains (DAPI)

• Compare with positive controls showing normal membranous distribution

• Evaluate at 400-600x magnification to clearly distinguish membrane boundaries from cytoplasmic regions

This distinction is particularly important when investigating the functional consequences of CDH1 missense variants, which may impair correct protein localization at the plasma membrane .

What are the most appropriate positive and negative controls for CDH1/E-cadherin immunofluorescence experiments?

Recommended Controls for CDH1/E-cadherin Immunofluorescence:

When analyzing samples with potential CDH1 mutations or methylation, include controls with known CDH1 alterations for comparison . This is particularly important when evaluating cancers with suspected but not confirmed CDH1 genetic or epigenetic alterations.

How can I optimize FITC-conjugated CDH1 antibody staining to detect low-level E-cadherin expression in research samples?

To optimize detection of low-level E-cadherin expression:

• Antigen retrieval optimization: Test multiple pH conditions (pH 6.0 citrate buffer vs. pH 9.0 EDTA buffer) and heating times to maximize epitope exposure without tissue damage

• Signal amplification: Consider tyramide signal amplification (TSA) which can enhance FITC signal 10-100 fold while maintaining localization specificity

• Antibody concentration titration: Perform systematic dilution series (1:50 to 1:500) to determine optimal signal-to-noise ratio

• Extended primary antibody incubation: Incubate at 4°C overnight rather than 1-2 hours at room temperature

• Blocking optimization: Use 5-10% normal serum from the species of secondary antibody origin plus 0.1-0.3% Triton X-100 and 1% BSA

• Confocal microscopy settings: Increase PMT gain and laser power while maintaining control of background; use spectral unmixing if autofluorescence is problematic

These approaches are particularly valuable when examining samples with CDH1 promoter methylation, which may lead to reduced but not completely absent protein expression .

What is the relationship between CDH1 genetic alterations and E-cadherin protein expression as detected by immunostaining?

The relationship between CDH1 genetic alterations and E-cadherin protein expression is complex:

• Truncating mutations (frameshift, nonsense) typically result in complete loss of E-cadherin expression detectable by immunostaining

• Missense mutations may cause:

  • Complete loss of expression

  • Reduced membrane expression with cytoplasmic accumulation

  • Near-normal membrane expression but with functional defects

• Promoter methylation generally results in reduced or absent expression (62.5% of cases with promoter methylation show complete E-cadherin loss)

• Large deletions involving 16q (CDH1 locus) frequently show loss of expression, especially when combined with promoter methylation of the remaining allele

Research has shown that 84% of invasive lobular carcinomas lacking CDH1 genetic alterations still show loss of E-cadherin expression, suggesting alternative inactivation mechanisms . For accurate interpretation, immunofluorescence results should be correlated with genetic and epigenetic analyses.

What are the key differences between using FITC-conjugated primary CDH1 antibodies versus unconjugated primary antibodies with FITC-conjugated secondary antibodies?

Comparison of Direct vs. Indirect Immunofluorescence for CDH1/E-cadherin Detection:

For critical analyses of E-cadherin expression in samples with potential CDH1 mutations, the indirect method is generally preferred for its signal amplification capabilities and better detection of reduced expression levels .

How can I accurately quantify E-cadherin membrane expression levels in cells with heterogeneous staining patterns?

For accurate quantification of heterogeneous E-cadherin membrane expression:

• Image acquisition standardization:

  • Use identical exposure settings across all samples

  • Capture multiple representative fields (minimum 5-10)

  • Employ z-stack imaging to capture complete membrane signal

• Quantification approaches:

  • Membrane-specific segmentation using specialized software (ImageJ with Membrane Plugin, CellProfiler)

  • Calculation of membrane-to-cytoplasm signal ratio rather than absolute intensity

  • Line profile analysis across cell-cell junctions

  • Co-localization coefficient with membrane markers

• Classification system:

  • Develop a scoring system (0, 1+, 2+, 3+) similar to HER2 scoring

  • Calculate H-score (percentage of positive cells × intensity score)

  • Report both percentage of membrane-positive cells and mean intensity

This approach is particularly important when evaluating E-cadherin in diffuse gastric cancers or lobular breast cancers with suspected CDH1 alterations, where heterogeneous expression patterns may reflect clonal evolution or second-hit inactivation mechanisms .

How should I interpret discordant results between CDH1 genetic testing and E-cadherin immunofluorescence findings?

When faced with discordant results between CDH1 genetic testing and E-cadherin immunofluorescence:

• Consider technical factors first:

  • Antibody specificity (validate with known controls)

  • Fixation artifacts (compare with other markers)

  • Tissue quality (evaluate morphology)

• Evaluate alternative mechanisms:

  • Epigenetic silencing through promoter methylation (occurs in 62.5% of cases without detectable mutations)

  • Post-transcriptional regulation (miRNAs)

  • Post-translational modifications affecting protein stability

  • Alterations in CDH1 regulatory elements outside sequenced regions

• Investigate related pathway components:

  • CTNNA1 mutations (α-catenin) can cause loss of E-cadherin membrane localization despite intact CDH1

  • Other adherens junction proteins (β-catenin, p120-catenin)

• Recommended follow-up studies:

  • CDH1 promoter methylation analysis (MSP or ddPCR)

  • mRNA expression analysis (qRT-PCR, RNA-seq)

  • Extended genetic analysis (whole exome sequencing)

  • Western blotting to confirm protein size and abundance

The molecular pathogenesis research suggests that approximately 37.5% of cases with typical lobular morphology and E-cadherin loss have neither CDH1 mutations nor promoter methylation, indicating additional mechanisms remain to be discovered .

How can FITC-conjugated CDH1 antibodies be utilized to study the functional consequences of CDH1 missense variants?

FITC-conjugated CDH1 antibodies are valuable tools for investigating functional consequences of CDH1 missense variants:

• Localization studies: Determine whether missense variants affect E-cadherin trafficking to the membrane using co-localization with organelle markers (ER, Golgi, endosomes)

• Dynamics analysis: Employ techniques like FRAP (Fluorescence Recovery After Photobleaching) to assess protein mobility and stability at adherens junctions

• Protein-protein interaction assessment: Combine with proximity ligation assay (PLA) to investigate interactions with key binding partners (β-catenin, p120-catenin, α-catenin)

• Structure-function correlation: Create a panel of cells expressing different CDH1 variants and analyze membrane localization patterns in relation to:

  • Variant location within protein domains

  • Conservation scores from in silico predictions

  • Clinical significance classification

Research has shown that pathogenic missense variants impair the correct binding of key adhesion-complex regulators and likely compromise normal E-cadherin localization and stability at the plasma membrane . Functional in vitro assays should be performed to evaluate the impact of CDH1 missense alterations on protein structure, trafficking, signaling, and E-cadherin function .

What are the best approaches for multiplexed detection of E-cadherin and other adherens junction proteins?

Optimal Multiplexing Strategies for E-cadherin and Adherens Junction Proteins:

When studying HDGC or lobular breast cancers, particularly important combinations include E-cadherin with α-catenin, as CTNNA1 mutations have been identified in CDH1-negative HDGC families . Additionally, analyzing the α-catenin and cytoplasmic E-cadherin phenotype can help identify potential CTNNA1-associated pathways .

How can I design experiments to investigate the second-hit inactivation mechanisms in CDH1 mutation carriers?

To investigate second-hit inactivation mechanisms in CDH1 mutation carriers:

• Tissue microdissection approach:

  • Microdissect E-cadherin-negative foci from FFPE sections of prophylactic gastrectomy specimens

  • Compare with adjacent normal mucosa showing normal E-cadherin expression

  • Analyze for:

    • Loss of heterozygosity (LOH) at 16q22.1 (CDH1 locus)

    • Promoter methylation of the remaining allele

    • Somatic mutations in the wild-type allele

• Single-cell analysis:

  • Perform laser capture microdissection of individual signet ring cells

  • Conduct single-cell genomic and epigenomic profiling

  • Compare multiple foci from the same patient to assess heterogeneity of second-hit mechanisms

• Temporal analysis in surveillance biopsies:

  • Collect serial endoscopic biopsies from CDH1 mutation carriers opting for surveillance

  • Track the emergence and evolution of second-hit events over time

  • Correlate with progression from indolent to clinically significant disease

• Molecular markers panel:

  • Develop a comprehensive panel including:

    • CDH1 promoter methylation (MSP or ddPCR)

    • 16q copy number analysis (FISH or CISH)

    • Targeted deep sequencing of CDH1

    • Expression analysis of E-cadherin and related proteins

This approach addresses the critical question of how long early lesions of HDGC can remain indolent until there is emergence of clinical disease . Continuing collection of data from patients who opt for endoscopic surveillance is essential to help answer this question, and a thorough analysis of second-hit inactivation mechanisms is necessary to define strategies for chemoprevention .

What are the most common causes of false negative E-cadherin staining and how can they be addressed?

Troubleshooting Guide for False Negative E-cadherin Staining:

When examining potential HDGC cases, false negative staining is particularly concerning as it may lead to misclassification. Always include positive control tissues on the same slide and consider dual-antibody approaches using antibodies targeting different E-cadherin domains .

How can I minimize photobleaching of FITC when imaging E-cadherin in thick tissue sections or during extended imaging sessions?

To minimize FITC photobleaching during extended E-cadherin imaging:

• Sample preparation optimization:

  • Add anti-fade mounting media containing radical scavengers (e.g., ProLong Gold, Vectashield)

  • Seal slide edges with nail polish to prevent oxygen penetration

  • Consider using oxygen-scavenging systems (glucose oxidase/catalase)

• Microscope settings adjustment:

  • Reduce excitation intensity to minimum required for adequate signal

  • Use neutral density filters to attenuate excitation light

  • Minimize exposure time and increase camera gain instead

  • Employ binning to reduce required light intensity

• Advanced imaging strategies:

  • Use resonant scanning in confocal microscopy to reduce pixel dwell time

  • Implement deconvolution to enhance signal post-acquisition

  • Consider alternative FITC derivatives with better photostability

  • Use computational approaches (RESTORE algorithm) to recover photobleached signal

• Alternative approaches for thick sections:

  • Consider optical clearing techniques compatible with immunofluorescence

  • Employ two-photon microscopy for deeper tissue penetration with less photobleaching

  • Use adaptive optics to maintain resolution at depth

These approaches are particularly important when imaging intramucosal signet ring cells in gastric tissue samples from CDH1 mutation carriers, where expert histopathological confirmation is recommended .

What strategies can address autofluorescence interference when using FITC-conjugated CDH1 antibodies in tissues with high natural fluorescence?

Strategies to address autofluorescence when using FITC-conjugated CDH1 antibodies:

• Pre-treatment methods:

  • Incubate sections in 0.1-1% sodium borohydride for 5-10 minutes before immunostaining

  • Treat with copper sulfate (10mM CuSO₄ in 50mM ammonium acetate buffer)

  • Apply Sudan Black B (0.1-0.3% in 70% ethanol) after immunostaining

• Optical approaches:

  • Use spectral unmixing to separate FITC signal from autofluorescence

  • Employ narrow bandpass filters to isolate FITC emission peak

  • Consider confocal microscopy with precisely defined spectral detection

• Alternative detection strategies:

  • Switch to longer wavelength fluorophores (Cy3, Alexa 555) less affected by tissue autofluorescence

  • Use Time-Gated Detection to separate long-lived autofluorescence from shorter-lived FITC signal

  • Implement Fluorescence Lifetime Imaging Microscopy (FLIM) to distinguish signals

• Computational correction:

  • Acquire autofluorescence signal from unstained serial sections

  • Digitally subtract from experimental images

  • Apply machine learning algorithms to identify and remove autofluorescence patterns

This issue is particularly relevant when examining gastric and breast tissue samples with high lipofuscin content, which can interfere with accurate assessment of E-cadherin expression patterns in HDGC and LBC diagnostic workups .