ACHE Antibody, FITC conjugated

What is acetylcholinesterase (ACHE) and what role does it play in biological systems?

Acetylcholinesterase (ACHE) is a 614-amino acid protein belonging to the Type-B carboxylesterase/lipase family. Its primary function is to hydrolyze the neurotransmitter acetylcholine rapidly at synaptic junctions, thereby terminating signal transduction at neuromuscular junctions . This enzymatic action prevents continuous stimulation of muscles and nerves, ensuring proper muscle contraction and cognitive processes . ACHE has predicted cellular localization in nuclear, membrane-associated, and secreted forms, with reported glycosylation sites . Beyond its classical role in neurotransmission, ACHE has been implicated in neuronal apoptosis processes , suggesting broader physiological relevance than previously recognized.

How do FITC-conjugated ACHE antibodies differ from unconjugated versions?

FITC (fluorescein isothiocyanate)-conjugated ACHE antibodies have the fluorescent FITC molecule directly attached to the antibody structure, providing immediate visualization capability without secondary detection reagents. Unlike unconjugated antibodies which require secondary antibody detection systems, FITC-conjugated antibodies:

• Allow direct detection in fluorescence-based applications

• Simplify experimental workflows by eliminating secondary antibody incubation steps

• Reduce background issues associated with secondary antibody cross-reactivity

• Enable multiplexing with other differently-conjugated primary antibodies

Commercial FITC-conjugated ACHE antibodies, such as those from Bioss Inc. and LifeSpan Biosciences, maintain target specificity while providing fluorescent detection capability .

What are the validated applications for FITC-conjugated ACHE antibodies?

FITC-conjugated ACHE antibodies have been specifically validated for fluorescence-based applications, with particular strength in cellular imaging techniques . The recommended dilutions should be optimized for each experimental system, as noted in product documentation stating "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" .

How can researchers validate the specificity of FITC-conjugated ACHE antibodies?

Validating antibody specificity is critical for ensuring reliable experimental results. For FITC-conjugated ACHE antibodies, comprehensive validation approaches include:

• Positive control tissues/cells: Use tissues known to express ACHE, such as mouse liver tissue or HEK-293 cells, which have been documented as positive controls .

• Knockout/knockdown validation: Compare staining patterns between wild-type samples and those with ACHE gene silencing.

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide (when available) to confirm signal reduction.

• Cross-validation with multiple antibodies: Use alternative antibodies targeting different ACHE epitopes and compare localization patterns.

• Correlation with functional assays: Compare antibody staining intensity with enzymatic activity measurements of ACHE.

The molecular weight for detection should be approximately 66-72 kDa, which corresponds to the observed molecular weight of ACHE in validated Western blot applications .

What considerations are important when designing multi-color flow cytometry experiments with FITC-conjugated ACHE antibodies?

When incorporating FITC-conjugated ACHE antibodies into multi-color flow cytometry panels, researchers should consider:

• Spectral overlap management: FITC emits in the green spectrum (peak ~525 nm), potentially overlapping with PE and other green-yellow fluorophores. Proper compensation is essential.

• Panel design strategy:

  • Reserve FITC for lower-expression targets if using brighter fluorophores like PE for other markers

  • Consider the relative abundance of ACHE in your sample when positioning in your panel

• Autofluorescence mitigation:

  • Include an unstained control for each tissue/cell type

  • Consider alternative conjugates for tissues with high green autofluorescence

• Sample preparation optimization:

  • Ensure complete red blood cell lysis when working with blood samples

  • Optimize fixation protocols to preserve both ACHE epitope and FITC fluorescence

• Titration is critical: As noted in product documentation, "Sample-dependent, Check data in validation data gallery" , suggesting that optimal concentration varies by application.

How can ACHE antibodies contribute to understanding neurodegenerative disease mechanisms?

ACHE antibodies provide valuable tools for investigating neurodegenerative disorders, particularly those with cholinergic system involvement:

• Alzheimer's Disease research applications:

  • Quantifying cholinergic neuron loss in brain regions

  • Evaluating the efficacy of acetylcholinesterase inhibitors

  • Studying ACHE interaction with amyloid-β peptides

• Parkinson's Disease investigations:

  • Examining non-motor symptoms related to cholinergic dysfunction

  • Monitoring autonomic nervous system changes

• Methodological approaches:

  • Immunohistochemistry of post-mortem brain tissue (validated dilution: 1:50-1:500)

  • Live-cell imaging in neuronal cultures

  • Multi-label co-localization with neurodegeneration markers

The ACHE protein's dual role in hydrolyzing acetylcholine and participating in neuronal apoptosis makes it particularly relevant for understanding disease progression mechanisms .

What is the optimal immunofluorescence protocol for FITC-conjugated ACHE antibodies?

Recommended Immunofluorescence Protocol:

• Sample preparation:

  • Fix cells/tissues with 4% paraformaldehyde (10-15 minutes at room temperature)

  • For tissue sections, use optimal cutting temperature (OCT) compound for cryosectioning

  • For paraffin sections, perform antigen retrieval with TE buffer pH 9.0 as recommended

• Permeabilization and blocking:

  • Permeabilize with 0.1-0.3% Triton X-100 in PBS (10 minutes)

  • Block with 5% normal serum (from same species as secondary antibody if using unconjugated primary) + 1% BSA in PBS (1 hour at room temperature)

• Antibody incubation:

  • Dilute FITC-conjugated ACHE antibody per manufacturer's recommendation (typically 1:50-1:500)

  • Incubate overnight at 4°C in a humidified chamber

  • Protect from light during and after antibody incubation

• Washing and counterstaining:

  • Wash 3× with PBS (5 minutes each)

  • Counterstain nuclei with DAPI (1:1000 in PBS, 5 minutes)

  • Mount with anti-fade mounting medium

• Imaging considerations:

  • Use appropriate filter sets for FITC (excitation ~495 nm, emission ~520 nm)

  • Capture negative controls with identical exposure settings

  • Minimize exposure time to prevent photobleaching

What controls are essential when working with FITC-conjugated ACHE antibodies?

Rigorous experimental design requires appropriate controls:

• Positive tissue controls: Include samples known to express ACHE such as:

  • Human/mouse liver tissue (validated in IHC)

  • Neuronal tissues or cell lines

  • Jurkat or HepG2 cells (validated in Western blot)

• Negative controls:

  • Isotype control: Use FITC-conjugated IgG of the same isotype (e.g., mouse IgG2a for antibodies like those in result )

  • Secondary-only control (for unconjugated primary experiments)

  • Peptide competition control (pre-incubation with immunizing peptide)

• Technical controls:

  • Unstained sample for autofluorescence assessment

  • Single-color controls for compensation in multi-color experiments

  • Concentration-matched controls when comparing different samples

• Biological validation:

  • ACHE inhibitor-treated samples (e.g., physostigmine) to correlate localization with activity

  • Genetic manipulation controls (siRNA, CRISPR) when available

How should samples be prepared to optimize ACHE antibody detection in different applications?

The documentation specifically notes for IHC applications: "suggested antigen retrieval with TE buffer pH 9.0; (*) Alternatively, antigen retrieval may be performed with citrate buffer pH 6.0" , highlighting the importance of optimization.

How can researchers address weak or absent signal when using FITC-conjugated ACHE antibodies?

When confronting weak fluorescence signal issues:

• Antibody concentration optimization:

  • Titrate antibody concentrations over a wider range (e.g., 1:10 to 1:1000)

  • Consider concentrated formats for challenging applications

• Sample preparation refinement:

  • Modify fixation duration (over-fixation can mask epitopes)

  • Test alternative antigen retrieval methods (switch between TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Increase permeabilization for intracellular targets

• Signal amplification strategies:

  • Consider biotin-streptavidin systems if signal strength is consistently problematic

  • Use anti-FITC secondary antibodies conjugated to brighter fluorophores

  • Implement tyramide signal amplification for very low abundance targets

• Imaging optimization:

  • Adjust exposure settings while avoiding autofluorescence

  • Use confocal microscopy for improved signal-to-noise ratio

  • Consider spectral unmixing for complex tissue autofluorescence

• Storage and handling issues:

  • FITC conjugates should be stored at -20°C and protected from light

  • Aliquot antibodies to avoid freeze-thaw cycles

  • Check for fluorophore degradation in older reagents

How should researchers quantify and analyze ACHE expression data from immunofluorescence experiments?

Quantitative analysis of ACHE immunofluorescence requires:

• Image acquisition standardization:

  • Capture all comparative samples with identical microscope settings

  • Include fluorescence intensity calibration standards

  • Maintain consistent exposure times across experimental groups

• Analysis approaches:

  • Intensity measurement: Mean fluorescence intensity (MFI) in defined regions

  • Distribution analysis: Nuclear vs. cytoplasmic vs. membrane localization

  • Co-localization quantification: Pearson's or Mander's coefficients with other markers

• Software tools recommendations:

  • ImageJ/FIJI with appropriate plugins for fluorescence quantification

  • CellProfiler for automated cell-by-cell analysis

  • Commercial platforms (Imaris, Volocity) for 3D analysis

• Normalization strategies:

  • Normalize to nuclear counterstain for cell density variations

  • Use housekeeping proteins for Western blot quantification

  • Include internal reference standards across experiments

• Statistical analysis:

  • Determine appropriate statistical tests based on data distribution

  • Account for technical and biological replicates appropriately

  • Consider power analysis for sample size determination

What are common sources of background and non-specific binding with FITC-conjugated antibodies and how can they be minimized?

Background issues can significantly impact data quality. Address them through:

• Blocking optimization:

  • Extend blocking time (1-2 hours at room temperature)

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Include 0.05-0.1% Tween-20 in blocking and antibody diluents

• Washing protocol enhancement:

  • Increase number of washes (5-6 times rather than standard 3)

  • Extend wash durations (10-15 minutes per wash)

  • Add 0.05-0.1% Tween-20 to wash buffers

• Autofluorescence management:

  • Pre-treat tissues with Sudan Black B (0.1-0.3% in 70% ethanol)

  • Use specialized autofluorescence quenching reagents

  • Implement spectral unmixing during image acquisition/analysis

• Antibody specificity verification:

  • Perform peptide competition controls

  • Test on samples known to be negative for ACHE

  • Consider alternative antibody clones if persistent cross-reactivity occurs

• FITC-specific considerations:

  • Protect samples from light throughout the protocol

  • Use fresh mounting media with anti-fade properties

  • Prepare working solutions immediately before use

How can FITC-conjugated ACHE antibodies be used in multiplexed imaging systems?

Multiplexed imaging with FITC-conjugated ACHE antibodies enables simultaneous visualization of multiple targets:

• Compatible fluorophore combinations:

  • FITC (green) pairs well with:

    • DAPI (blue) for nuclear counterstaining

    • Cy3/TRITC (red) for second target

    • Cy5/APC (far-red) for third target

• Sequential staining approach:

  • Begin with FITC-conjugated ACHE antibody

  • Follow with additional unconjugated primary antibodies

  • Complete with spectrally distinct secondary antibodies

• Technical considerations:

  • Account for spectral bleed-through during image acquisition

  • Implement proper controls for each channel

  • Consider linear unmixing for closely overlapping fluorophores

• Novel multiplexing technologies integration:

  • Cyclic immunofluorescence for high-parameter imaging

  • Mass cytometry adaptation using metal-tagged antibodies

  • Super-resolution microscopy compatibility assessment

The FITC conjugate's excitation/emission characteristics (approximately 495/520 nm) provide excellent separation from red and far-red fluorophores, making it ideal for multiplexed imaging systems .

What insights can ACHE antibodies provide in cancer research applications?

ACHE antibodies have emerging applications in oncology research:

• Cancer-specific observations:

  • "AChE activity is significantly lower in aspirates from squamous cell carcinomas" , suggesting altered cholinergic signaling in cancer

• Research applications:

  • Evaluating non-neuronal cholinergic system alterations in tumors

  • Investigating ACHE as a potential biomarker for specific cancer types

  • Studying relationships between ACHE expression and cancer cell proliferation/migration

• Methodological approaches:

  • Immunohistochemistry of tumor tissue microarrays

  • Flow cytometric analysis of circulating tumor cells

  • Correlation of ACHE levels with treatment response

• Recommended experimental models:

  • Human cancer cell lines (validated examples include SkHep1, Snu387, SW620)

  • Patient-derived xenografts

  • Clinical tumor specimens with matched normal tissues

This research direction represents an expanding frontier beyond traditional neuroscience applications of ACHE antibodies.