IMMT Antibody

What is IMMT and why is it important in cellular research?

IMMT (inner membrane protein, mitochondrial), also known as mitofilin, is a critical component of the mitochondrial contact site and cristae organizing system (MICOS). This protein plays an essential role in maintaining inner mitochondrial membrane architecture and crista junction formation . IMMT has a calculated molecular weight of 90 kDa but is typically observed at 80-90 kDa in experimental conditions .

Research significance extends beyond basic mitochondrial biology, as IMMT expression has been associated with clinical outcomes in several cancers. High IMMT expression correlates with poorer prognosis in lung adenocarcinoma patients, correlating significantly with advanced disease stage, larger tumor size, and intratumoral vascular invasion . Similar prognostic value has been identified in breast cancer, where IMMT serves as an independent diagnostic biomarker associated with advanced clinical status and poor relapse-free survival .

Genetic studies have demonstrated that IMMT ablation induces lethal disruption of the MICOS complex, highlighting its fundamental importance in cellular viability .

How do I select the most appropriate IMMT antibody for my research application?

Selection of an appropriate IMMT antibody should follow a systematic approach based on your specific experimental application and target species:

• Define your application requirements: Different applications (WB, IHC, IF/ICC, FC, IP) require antibodies validated specifically for those techniques. For example, antibody 10179-1-AP has been validated for multiple applications including WB (1:5000-1:50000 dilution), IHC (1:50-1:500), and IF/ICC (1:50-1:500) .

• Consider species reactivity: Verify that the antibody has been validated in your species of interest. The IMMT antibody 10179-1-AP shows reactivity with human, mouse, and rat samples, with cited reactivity extending to hamster models .

• Utilize antibody search resources: Several databases can help identify validated antibodies:

  • Antibodypedia for validated antibodies and antigens

  • The Antibody Registry for unique identifiers

  • CiteAb for citation-ranked antibodies

• Review validation data: Examine the manufacturer's validation data and, when possible, published literature using the antibody. Look for evidence of specificity such as:

  • Single band at expected molecular weight (80-90 kDa for IMMT)

  • Absence of signal in knockout/knockdown samples

  • Consistent localization pattern (mitochondrial for IMMT)

• Consider antibody format: Polyclonal antibodies often provide higher sensitivity but potentially lower specificity compared to monoclonals. The antibody 10179-1-AP is a rabbit polyclonal with IgG isotype .

Always verify antibody performance in your specific experimental system, as reactivity can vary based on sample preparation, fixation methods, and protein expression levels.

What are the critical differences between monoclonal and polyclonal IMMT antibodies?

The choice between monoclonal and polyclonal IMMT antibodies depends on your experimental goals and requirements:

Polyclonal IMMT Antibodies:

• Recognize multiple epitopes on the IMMT protein, potentially increasing detection sensitivity

• Provide more robust detection across different applications and conditions

• May show greater tolerance to minor protein denaturation or modifications

• Example: The rabbit polyclonal antibody 10179-1-AP demonstrates versatility across multiple applications (WB, IHC, IF/ICC, FC, IP)

• Potential drawback: May exhibit batch-to-batch variability and higher background in some applications

Monoclonal IMMT Antibodies:

• Recognize a single epitope with high specificity

• Provide consistent performance with minimal batch variation

• Particularly valuable for distinguishing between closely related protein isoforms

• Potential drawback: May be more sensitive to epitope masking by fixation or denaturation

When selecting between these formats, consider:

• Application requirements: For quantitative studies or where high specificity is critical, monoclonals may be preferred

• Sample processing: If samples undergo variable fixation or processing methods, polyclonals may provide more consistent detection

• Background concerns: In tissues with high autofluorescence or endogenous peroxidase activity, the higher specificity of monoclonals may be advantageous

For critical experiments, validation with both antibody types or with antibodies targeting different epitopes (Independent antibody validation) can provide enhanced confidence in results .

What dilution ranges should I test when optimizing IMMT antibody for Western blotting?

Optimizing IMMT antibody dilution for Western blotting requires systematic testing to balance signal strength against background. Based on established protocols and manufacturer recommendations:

• Start with recommended ranges: For antibody 10179-1-AP, the manufacturer recommends a broad initial range of 1:5000-1:50000 for Western blotting . This wide range suggests high sensitivity and requires careful optimization.

• Perform a dilution series: Begin with 3-4 dilutions within the recommended range:

  • High concentration: 1:5000

  • Medium concentration: 1:15000

  • Low concentration: 1:30000

  • Very low concentration: 1:50000

• Evaluate signal-to-noise ratio: The optimal dilution provides clear detection of the 80-90 kDa IMMT band with minimal background. Remember that:

  • Too concentrated antibody increases background and reduces specificity

  • Too dilute antibody may result in weak or undetectable signal

• Consider sample type: Different sample types may require different optimal dilutions:

  • Cell lines with high IMMT expression (like HeLa or HEK-293) may work well with higher dilutions

  • Tissue samples may require more concentrated antibody, especially for low-abundance proteins

• Loading control optimization: Simultaneously optimize loading control antibodies to ensure proper normalization for quantitative analysis

Document your optimization process systematically, as these findings may be required for publication to satisfy reproducibility requirements . Once optimized, maintain consistent conditions across experimental replicates, including antibody dilution, incubation time, temperature, and washing steps.

How should I properly validate IMMT antibody specificity for immunohistochemistry applications?

Validating IMMT antibody specificity for immunohistochemistry (IHC) requires a multi-step approach to ensure reliable and reproducible results:

• Positive controls: Use tissues or cells known to express IMMT:

  • Human breast cancer tissue has been validated for antibody 10179-1-AP

  • Cell lines such as HeLa cells that express IMMT can serve as positive controls

  • Include standardized samples with each experimental run

• Negative controls: Implement multiple strategies to verify specificity:

  • Primary antibody omission: Process sections identically but omit primary antibody to assess secondary antibody specificity

  • Knockout/knockdown controls: Ideally, use IMMT-knockout tissues or CRISPR/Cas9-mediated knockout cells

  • Blocking peptide control: Pre-incubate antibody with excess immunizing peptide/protein to confirm signal specificity

• Antigen retrieval optimization: IMMT antibody 10179-1-AP requires specific retrieval conditions:

  • Recommended: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Test both methods to determine optimal conditions for your tissue type

• Dilution optimization: Test a range of dilutions (1:50-1:500 for 10179-1-AP)

• Independent antibody validation: Confirm staining pattern with a second antibody targeting a different IMMT epitope:

  • Matching patterns from independent antibodies significantly increases confidence

  • This approach meets enhanced validation criteria as defined by validation guidelines

• Orthogonal validation: Compare protein expression by IHC with mRNA expression data:

  • High or medium consistency between protein and RNA expression patterns increases confidence

  • This comparison serves as a basis for enhanced validation status

Document all validation steps thoroughly for publication, as journals increasingly require demonstration of antibody specificity, especially for studies focused on protein expression patterns or localization .

What are the optimal fixation and permeabilization conditions for IMMT immunofluorescence?

Optimizing fixation and permeabilization conditions is critical for accurate IMMT detection by immunofluorescence, as these steps affect epitope accessibility and mitochondrial membrane preservation:

Fixation Recommendations:

• Paraformaldehyde (PFA) fixation:

  • 4% PFA for 15-20 minutes at room temperature preserves mitochondrial morphology

  • Avoid overfixation as it can mask epitopes in the mitochondrial inner membrane

  • PFA is compatible with IMMT antibody 10179-1-AP as validated in HeLa cells

• Methanol fixation alternative:

  • Ice-cold 100% methanol for 5-10 minutes at -20°C

  • May provide superior detection of some mitochondrial membrane proteins

  • Test alongside PFA to determine optimal conditions for your specific IMMT antibody

Permeabilization Options:

• For PFA-fixed samples:

  • 0.1-0.2% Triton X-100 in PBS for 10 minutes at room temperature

  • Alternative: 0.1% saponin for more gentle permeabilization that better preserves membrane structures

• For methanol-fixed samples:

  • Additional permeabilization is typically unnecessary as methanol serves as both fixative and permeabilizing agent

Optimization Considerations:

• Cell type-specific adjustments:

  • Adherent cell lines (HeLa, HEK-293) work well with standard protocols

  • Primary cells may require gentler permeabilization

  • Tissue sections require optimization of antigen retrieval methods

• Co-staining compatibility:

  • When co-staining with other mitochondrial markers, ensure fixation methods are compatible with all antibodies

  • Consider using mitochondrial markers like TOM20 (outer membrane) alongside IMMT to verify mitochondrial localization

• Blocking conditions:

  • 5-10% normal serum (from the species of secondary antibody) for 1 hour

  • Addition of 0.1-0.3% Triton X-100 to blocking solution may improve antibody penetration

Systematic testing of these conditions is recommended, with documentation of optimization parameters for publication and reproducibility. The recommended dilution range for IMMT antibody 10179-1-AP in immunofluorescence applications is 1:50-1:500 .

What are the essential controls required for validating IMMT antibody experiments?

Implementing comprehensive controls is essential for ensuring the reliability and reproducibility of IMMT antibody experiments. Based on established guidelines, the following controls should be considered:

Primary Antibody Controls (Specificity):

• Genetic knockdown/knockout: The gold standard control demonstrating antibody specificity is testing in IMMT-knockout or CRISPR/Cas9-edited samples, which should show absence of specific signal

• Antigen competition: Pre-incubating the antibody with excess IMMT peptide/protein should eliminate specific staining

• Independent antibodies: Using multiple antibodies targeting different IMMT epitopes that produce consistent results provides strong validation

Secondary Antibody Controls:

• No primary antibody: Samples processed identically but omitting the primary IMMT antibody to identify non-specific secondary antibody binding

• Isotype control: Using non-immune IgG from the same species as the primary antibody at equivalent concentration

• Cross-reactivity test: When performing multi-label experiments, controls to ensure secondary antibodies don't cross-react with non-target primary antibodies

Label Controls:

• Autofluorescence/endogenous enzyme control: Samples processed without primary and secondary antibodies to identify endogenous signal

• Fluorophore/enzyme substrate controls: Necessary when changing detection systems or working with new tissue types

Control Selection Based on Application:

These controls should be systematically implemented and documented according to the "Minimum Requirements" and "Supplementary Recommendations" framework outlined in research guidelines . For publication, detailed description of the controls performed is essential to demonstrate experimental rigor and facilitate reproducibility.

How can I confirm that my IMMT antibody is recognizing the correct protein in my experimental system?

Confirming that your IMMT antibody is recognizing the correct protein requires a multi-faceted approach applying several validation strategies:

Molecular Weight Verification:

• IMMT has a calculated molecular weight of 90 kDa but is typically observed at 80-90 kDa in experimental conditions

• Verify that your Western blot shows a band within this expected range

• Be cautious of additional bands that may represent isoforms, degradation products, or non-specific binding

Genetic Manipulation Approaches:

• siRNA knockdown: Transfect cells with IMMT-specific siRNA and confirm reduced signal intensity that correlates with knockdown efficiency

• CRISPR/Cas9 knockout: Generate IMMT knockout cell lines as the definitive negative control

• Overexpression: Transfection with IMMT expression vectors should produce increased signal intensity

Orthogonal Method Validation:

• Compare protein expression detected by antibody with mRNA expression data

• High or medium consistency between antibody staining patterns and RNA expression levels provides enhanced validation

• Correlation with mass spectrometry data, if available, provides strong cross-platform confirmation

Independent Antibody Validation:

• Test multiple antibodies targeting different IMMT epitopes

• Consistent results between independent antibodies significantly increases confidence

• This approach meets criteria for enhanced validation as defined by validation guidelines

Cellular Localization Assessment:

• IMMT should show distinctive mitochondrial localization in immunofluorescence experiments

• Co-localization with established mitochondrial markers (e.g., MitoTracker or other mitochondrial proteins) provides further confirmation

• Positive IF/ICC detection has been validated in HeLa cells using antibody 10179-1-AP

Positive Control Tissues/Cells:

• Include samples known to express IMMT:

  • HeLa, HEK-293, HepG2, COLO 320, Raji, and MCF-7 cell lines

  • Mouse and rat brain tissue have been validated for antibody 10179-1-AP

Document all validation approaches systematically, as this information will strengthen published findings and satisfy increasingly stringent journal requirements for antibody validation .

What validation approaches are required to meet the "Enhanced" validation status for IMMT antibodies?

Achieving "Enhanced" validation status for IMMT antibodies requires implementing stringent validation approaches that definitively confirm antibody specificity. According to established validation frameworks , the following criteria must be met:

Qualifying for Enhanced Validation Status:

• Orthogonal Validation:

  • Correlation between antibody-based detection and an antibody-independent method

  • Compare IMMT protein levels detected by immunohistochemistry with mRNA expression data

  • High or medium consistency between protein and RNA expression patterns is required

  • This approach verifies that the antibody recognizes the intended target by an independent method

• OR Independent Antibody Validation:

  • Testing multiple antibodies targeting different IMMT epitopes

  • Antibodies must show highly similar staining patterns/results

  • The exact epitope or binding region of each antibody must be documented

  • This approach provides confidence that consistent results from different antibodies verify the correct target

Documentation Requirements:

To achieve Enhanced validation status, detailed documentation must include:

• Specific validation strategy employed (Orthogonal or Independent Antibody)

• Complete experimental details of validation procedures

• Raw data showing correlation between methods

• For Independent Antibody validation: documentation of epitope regions for each antibody

Validation Reliability Scoring:

Based on the reliability scoring system :

Implementing these validation strategies not only establishes Enhanced validation status but also significantly increases confidence in experimental results and supports reproducibility across different research groups. The enhanced validation framework represents the current gold standard in antibody validation and is increasingly expected by high-impact journals .

How can IMMT antibodies be applied to study mitochondrial dynamics and cristae organization?

IMMT antibodies serve as powerful tools for investigating mitochondrial dynamics and cristae organization, offering insights into both normal physiological processes and disease states:

Advanced Microscopy Applications:

• Super-resolution microscopy:

  • IMMT antibodies combined with techniques such as STED or STORM microscopy can reveal the precise arrangement of MICOS complex components at crista junctions

  • Co-staining with outer membrane markers (TOM20) and inner membrane markers can elucidate IMMT's role in membrane contact sites

  • Use dilution ranges of 1:50-1:500 for optimal signal with minimal background

• Live-cell imaging approaches:

  • Expression of fluorescently-tagged IMMT can complement antibody studies

  • Photobleaching experiments (FRAP) can assess IMMT mobility and structural roles

  • Correlative light-electron microscopy using IMMT antibodies can connect protein localization with ultrastructural features

Multi-omics Investigation Strategies:

• Isolation of mitochondria-associated complexes:

  • IMMT antibodies can be used for immunoprecipitation (IP) of MICOS components

  • Recommended protocol: 0.5-4.0 μg antibody for 1.0-3.0 mg total protein lysate

  • Co-IP experiments reveal interaction partners and complex composition

• Combined genomic and proteomic approaches:

  • Integrating IMMT antibody data with RNA-seq and mass spectrometry provides multi-level insights

  • Research has demonstrated the value of this approach in identifying IMMT's role in cancer progression

Disease-Relevant Applications:

• Cancer research applications:

  • IMMT expression serves as a prognostic marker in lung adenocarcinoma and breast cancer

  • Immunohistochemistry protocols (1:50-1:500 dilution) can assess IMMT levels in patient samples

  • Flow cytometry (0.20 μg per 10^6 cells) can quantify IMMT expression in circulating tumor cells

• Neurodegenerative disease models:

  • IMMT antibodies can reveal mitochondrial structural abnormalities in disease models

  • Mouse and rat brain tissues have been validated as positive controls for antibody 10179-1-AP

When designing these advanced applications, incorporate appropriate controls (as detailed in question 3.1) and consider employing the enhanced validation approaches (question 3.3) to ensure reliable interpretation of results. Combining multiple techniques provides the most comprehensive understanding of IMMT's role in mitochondrial structure and function.

What approaches can resolve contradictory IMMT antibody staining patterns between immunofluorescence and Western blot?

Resolving contradictions between IMMT immunofluorescence (IF) and Western blot (WB) results requires systematic investigation of technical and biological factors that could explain the discrepancies:

Technical Factors to Investigate:

• Epitope accessibility differences:

  • Protein conformation differs between applications: WB uses denatured protein while IF often detects native conformation

  • Solution: Test antibodies known to recognize linear (denaturation-resistant) epitopes for WB and conformation-sensitive antibodies for IF

  • Antibodies that recognize native epitopes often do not interact with denatured epitopes

• Fixation and processing effects:

  • Overfixation can mask IMMT epitopes, particularly in the mitochondrial inner membrane

  • Solution: Test multiple fixation protocols (4% PFA, methanol, acetone) and antigen retrieval methods

  • For antibody 10179-1-AP, suggested retrieval uses TE buffer pH 9.0 or citrate buffer pH 6.0

• Antibody validation status:

  • Check if the antibody is validated for both applications

  • Antibody 10179-1-AP is validated for both WB (1:5000-1:50000) and IF (1:50-1:500)

  • Consider using an antibody with "Enhanced" validation status

Biological Explanations to Consider:

• Post-translational modifications:

  • Phosphorylation, ubiquitination, or other modifications may affect epitope recognition

  • These modifications can differ between experimental conditions or cell states

  • "Reduced antibody signals inferring reduced protein levels may be due to changes in posttranslational modification and not due to actual reduced levels of the protein amount"

• Protein isoforms or splice variants:

  • Different IMMT isoforms may be present in different cellular compartments

  • Solution: Use isoform-specific antibodies or RNA analysis to identify which isoforms are expressed

• Protein complexes and interactions:

  • IMMT's participation in the MICOS complex may mask epitopes in native conditions

  • Solution: Compare results using mild detergents versus stronger denaturing conditions

Resolution Strategy:

• Orthogonal validation approach:

  • Implement genetic manipulation (siRNA knockdown or CRISPR knockout of IMMT)

  • Both IF and WB signals should decrease proportionally with knockdown

  • This test can determine which technique is giving correct results

• Independent antibody verification:

  • Use multiple antibodies targeting different IMMT epitopes

  • Consistent results between independent antibodies significantly increases confidence

• Method optimization:

  • Systematically vary antibody concentration, incubation conditions, and detection methods

  • Document optimization parameters thoroughly for reproducibility

When reporting results, transparently discuss any discrepancies and the approaches taken to resolve them, which strengthens the rigor of the research and provides valuable methodological insights for the scientific community .

How can IMMT antibodies be properly utilized in studies of mitochondrial dysfunction in disease models?

Utilizing IMMT antibodies effectively in disease model studies requires careful experimental design and integration with complementary approaches to provide comprehensive insights into mitochondrial dysfunction:

Experimental Design Considerations:

• Appropriate model selection:

  • Cell models: Validated cell lines include HeLa, HEK-293, COLO 320, and MCF-7

  • Animal models: Significant research has utilized mouse and rat models

  • Patient samples: IMMT antibodies have been validated in human cancer tissues

• Temporal analysis:

  • Implement time-course studies to track IMMT expression changes during disease progression

  • For acute interventions, establish appropriate timepoints based on mitochondrial dynamics (typically 24-72 hours)

• Quantitative approaches:

  • Western blot: Use dilution range 1:5000-1:50000 with proper loading controls

  • Flow cytometry: Use 0.20 μg per 10^6 cells in 100 μl suspension for precise quantification

  • Always include standard curves with recombinant protein for absolute quantification when possible

Disease-Specific Applications:

• Cancer research applications:

  • IHC protocols (1:50-1:500) for patient tumor samples

  • Prognostic value assessment: High IMMT expression correlates with poor prognosis in lung adenocarcinoma and breast cancer

  • Multivariate analysis has established IMMT as an independent predictor of survival (HR, 1.99; 95% CI, 1.06–3.74)

• Neurodegenerative disease models:

  • IMMT antibodies can assess mitochondrial structural integrity in neuronal models

  • Combine with synaptic markers to study mitochondrial distribution at synapses

  • Mouse and rat brain tissues serve as validated positive controls

• Genetic disorders affecting mitochondria:

  • IMMT genetic ablation has been shown to induce lethal disruption of MICOS

  • Antibodies can assess partial loss or mislocalization in patient samples

When designing these studies, implement the comprehensive validation strategies described in previous sections to ensure reliable interpretation of results. Document all experimental parameters thoroughly to enable reproducibility and translational relevance of findings.

What are the most common causes of non-specific binding with IMMT antibodies and how can they be addressed?

Non-specific binding with IMMT antibodies can compromise experimental results, but systematic troubleshooting can identify and resolve these issues:

Common Causes and Solutions:

• Insufficient blocking:

  • Problem: Inadequate blocking allows antibodies to bind non-specifically to charged surfaces

  • Solution: Optimize blocking conditions:

    • Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

    • Test different blocking agents (5-10% normal serum from secondary antibody species, BSA, non-fat dry milk)

    • For IF applications, add 0.1-0.3% Triton X-100 to blocking solution to reduce membrane-associated background

• Excessive antibody concentration:

  • Problem: Too concentrated primary antibody increases non-specific interactions

  • Solution: Perform systematic dilution series

    • For Western blot: Test range from 1:5000 to 1:50000

    • For IHC/IF: Test range from 1:50 to 1:500

    • Document optimal concentration that maximizes specific signal while minimizing background

• Cross-reactivity with related proteins:

  • Problem: Antibodies may recognize proteins with homologous domains

  • Solution: Validate with genetic knockdown/knockout controls

    • CRISPR/Cas9-mediated knockout serves as definitive control

    • Compare with Independent antibody targeting different epitope

    • Consider pre-absorbing antibody with related proteins if cross-reactivity is identified

• Sample preparation issues:

  • Problem: Incomplete fixation or over-fixation can expose non-specific binding sites

  • Solution: Optimize fixation protocol

    • For IHC: Test both TE buffer pH 9.0 and citrate buffer pH 6.0 for antigen retrieval

    • For IF: Compare 4% PFA (10-20 min) with methanol fixation

    • Fresh samples typically yield better results than archived materials

Application-Specific Considerations:

Advanced Troubleshooting Methods:

• Antibody purification:

  • If high background persists, consider pre-absorbing antibody against fixed cells/tissues lacking IMMT

  • For 10179-1-AP, which is already antigen affinity purified , additional purification is rarely necessary

• Signal amplification alternatives:

  • For low abundance targets, consider enzymatic amplification systems (TSA) or more sensitive detection methods

  • Balance increased sensitivity with potential for increased background

• Comparison with genetic approaches:

  • Correlate antibody staining patterns with fluorescently tagged IMMT expression

  • This provides independent verification of localization patterns

Always document optimization procedures thoroughly, as these details are critical for reproducibility and may be required for publication. When persistent non-specific binding occurs despite optimization, consulting with the antibody manufacturer or switching to an alternative validated antibody may be necessary.

How can I optimize IMMT antibody conditions for dual immunofluorescence with other mitochondrial markers?

Optimizing dual immunofluorescence with IMMT antibodies and other mitochondrial markers requires careful consideration of antibody compatibility, protocol harmonization, and signal separation:

Antibody Selection and Compatibility:

• Host species considerations:

  • Choose primary antibodies raised in different host species to avoid cross-reactivity

  • IMMT antibody 10179-1-AP is rabbit-derived , so pair with mouse, goat, or rat antibodies for other markers

  • Common compatible pairs:

    • Rabbit anti-IMMT + Mouse anti-TOM20 (outer membrane)

    • Rabbit anti-IMMT + Mouse anti-ATP5A (matrix)

• Isotype selection:

  • If using antibodies from the same species is unavoidable, use different isotypes and isotype-specific secondaries

  • Sequential staining with complete blocking between steps can also minimize cross-reactivity

Protocol Harmonization:

• Fixation optimization:

  • Different mitochondrial proteins may require different fixation methods

  • Test both 4% PFA (15-20 min) and methanol fixation to identify conditions compatible with all antibodies

  • Document optimal conditions that preserve epitopes for both IMMT and partner proteins

• Antigen retrieval balancing:

  • For tissue sections, test both recommended retrieval methods for IMMT (TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Identify retrieval conditions that work for all antibodies in the multiplex panel

• Blocking and permeabilization:

  • Use blocking serum corresponding to both secondary antibody species

  • Optimize Triton X-100 concentration (0.1-0.3%) to ensure adequate permeabilization without destroying mitochondrial membrane integrity

Antibody Dilution and Signal Optimization:

• Sequential dilution optimization:

  • First optimize each antibody individually

  • For IMMT antibody 10179-1-AP, test 1:50, 1:100, 1:250, and 1:500 dilutions

  • Then test in combination, adjusting as needed to balance signal intensities

• Signal separation strategies:

  • Choose fluorophores with minimal spectral overlap

  • For confocal microscopy, perform sequential scanning rather than simultaneous acquisition

  • Consider linear unmixing for fluorophores with partial spectral overlap

• Controls for dual staining:

  • Single-stained controls to establish signal specificity and bleed-through

  • Secondary-only controls for each channel

  • Absorption controls (primary antibody pre-incubated with antigen)

Advanced Optimization Approaches:

• Signal amplification balancing:

  • If signal intensities differ significantly between markers, consider TSA amplification for the weaker signal

  • Adjust exposure settings to balance visualization of both markers

• Specialized approaches for challenging samples:

  • For high autofluorescence tissues, consider Sudan Black B treatment or spectral imaging

  • For thick sections, optimize clearing protocols compatible with antibody epitopes

• Three-dimensional analysis:

  • For 3D reconstruction, collect z-stacks with optimal step size

  • Use appropriate software for colocalization analysis and quantification

Example Optimized Protocol:

• Fix cells in 4% PFA for 15 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 for 10 minutes

• Block with 5% normal goat serum + 5% normal donkey serum for 1 hour

• Incubate with rabbit anti-IMMT (1:100) and mouse anti-TOM20 (1:250) overnight at 4°C

• Wash 3×10 minutes with PBS + 0.1% Tween-20

• Incubate with goat anti-rabbit Alexa Fluor 488 and donkey anti-mouse Alexa Fluor 594 (both 1:500) for 1 hour

• Counterstain nuclei with DAPI (1:1000)

• Mount in anti-fade medium

This optimized approach enables reliable colocalization analysis to understand IMMT's relationship with other mitochondrial components in normal and pathological conditions.

What strategies can minimize variability between replicate experiments using IMMT antibodies?

Minimizing variability between replicate experiments with IMMT antibodies requires implementing standardized protocols, rigorous quality control, and systematic documentation:

Protocol Standardization:

• Detailed standard operating procedures (SOPs):

  • Create comprehensive SOPs for each application (WB, IHC, IF, FC)

  • Document all critical parameters including:

    • Sample preparation methods

    • Buffer compositions with exact pH values

    • Incubation times and temperatures

    • Exact antibody dilutions (e.g., 1:10000 for WB rather than range 1:5000-1:50000)

• Reagent consistency:

  • Use antibodies from the same lot when possible (record lot numbers)

  • Prepare large batches of buffers and store aliquots

  • Standardize protein extraction and quantification methods

  • Implement consistent blocking reagents (source, concentration, preparation)

• Equipment calibration:

  • Regularly calibrate and maintain all equipment (pipettes, pH meters, imaging systems)

  • Document equipment settings for image acquisition

  • Use consistent exposure settings for fluorescence imaging

Quality Control Measures:

• Internal controls in every experiment:

  • Include positive control samples (HeLa cells, HEK-293 cells, validated tissues)

  • Run negative controls (knockout samples when available)

  • Use reference standards for quantitative experiments

  • Include housekeeping protein controls (beta-actin, GAPDH) for loading normalization

• Antibody validation checks:

  • Periodically reverify antibody performance

  • Test new lots against previous lots before implementation

  • Store antibodies according to manufacturer recommendations with minimal freeze-thaw cycles

• Statistical approaches:

  • Determine appropriate sample sizes through power analysis

  • Process samples in random order to avoid batch effects

  • Implement blinding where appropriate for analysis

Practical Implementation Strategies:

• Batch processing:

  • Process all experimental samples in parallel when possible

  • For large experiments, distribute conditions across multiple batches rather than processing by condition

  • Include inter-batch calibration samples

• Timing consistency:

  • Standardize sample collection timing

  • Maintain consistent incubation times (use timers)

  • Process all samples from collection to analysis within similar timeframes

• Temperature management:

  • Control laboratory temperature

  • Monitor refrigerator/freezer temperatures

  • Pre-equilibrate reagents to appropriate temperature before use

Documentation and Reporting:

• Comprehensive experimental records:

  • Document all experimental conditions in laboratory notebooks

  • Record any deviations from SOPs

  • Note batch numbers of all reagents

  • Preserve original unprocessed images

• Metadata capture:

  • Record all instrument settings

  • Document image processing steps

  • Maintain raw quantification data alongside analyzed results

• Reporting for publication:

  • Provide detailed methods including antibody information:

    • Catalog number and RRID (AB_2127193 for 10179-1-AP)

    • Lot number

    • Dilution used and incubation conditions

    • Validation method

Example Standardization Strategy for Western Blot:

• Extract proteins using standardized RIPA buffer with protease inhibitors

• Quantify by BCA method in triplicate

• Load 20 μg protein per lane

• Include recombinant IMMT protein standard curve

• Transfer using specific conditions (25V for 16 hours at 4°C)

• Block with 5% non-fat milk in TBST for exactly 1 hour at room temperature

• Incubate with IMMT antibody 10179-1-AP at 1:10000 overnight at 4°C

• Wash 3×10 minutes with TBST

• Develop using standardized ECL reagent with fixed exposure time

• Analyze band intensity using validated software with consistent quantification parameters

Implementing these strategies systematically will significantly reduce inter-experimental variability and enhance the reproducibility of results across different operators and laboratories.

How can computational approaches improve IMMT antibody specificity prediction and design?

Computational approaches are revolutionizing antibody specificity prediction and design, with particular relevance for complex targets like IMMT. Recent advances offer powerful tools for researchers:

Advanced Specificity Prediction Methods:

• Biophysics-informed modeling:

  • Models trained on experimentally selected antibodies can identify distinct binding modes associated with specific antigens

  • This approach enables "the prediction and generation of specific variants beyond those observed in the experiments"

  • Particularly valuable for discriminating between chemically similar epitopes, which is essential for IMMT-specific detection

• Energy function optimization:

  • Computational approaches can "optimize over s the energy functions E associated with each mode sw w"

  • For IMMT-specific antibodies, this allows:

    • Design of cross-specific antibodies interacting with multiple IMMT epitopes

    • Design of highly specific antibodies targeting single epitopes while excluding others

• Sequence-structure relationship analysis:

  • Combining antibody sequence data with structural information about IMMT epitopes

  • Prediction algorithms can identify key residues determining specificity

  • Machine learning models trained on existing antibody-antigen interaction data improve prediction accuracy

Practical Applications for IMMT Research:

• Custom specificity profile design:

  • "Our model can be employed to design novel antibody sequences with predefined binding profiles"

  • Potential to design IMMT antibodies that:

    • Discriminate between IMMT isoforms

    • Specifically recognize post-translationally modified IMMT

    • Target IMMT only when incorporated in specific protein complexes

• Experimental-computational feedback loops:

  • Iterative approach combining computational prediction with experimental validation

  • "The combination of biophysics-informed modeling and extensive selection experiments holds broad applicability"

  • For IMMT research, this can accelerate development of highly specific research tools

• Cross-reactivity minimization:

  • Computational screening against the proteome to identify potential cross-reactivities

  • Design modifications to enhance IMMT specificity while reducing off-target binding

  • Particularly valuable for discriminating IMMT from other mitochondrial membrane proteins

Future Directions and Emerging Technologies:

• Integration with high-throughput experimental methods:

  • Combining computational prediction with phage display selection

  • "We conducted a series of phage display experiments involving antibody selection against diverse combinations of closely related ligands"

  • Applying similar approaches to IMMT epitopes could yield highly specific antibodies

• AI-enhanced epitope mapping:

  • Deep learning models can predict optimal IMMT epitopes for antibody generation

  • AlphaFold2 and similar protein structure prediction tools can model IMMT-antibody interactions

  • These approaches may identify novel epitopes not accessible by traditional methods

• Antibody optimization beyond selection:

  • Computational maturation of existing IMMT antibodies to enhance:

    • Specificity for particular applications

    • Affinity under specific experimental conditions

    • Performance across diverse species for comparative studies

The integration of these computational approaches with traditional antibody generation technologies promises to yield IMMT antibodies with unprecedented specificity and customized binding properties, advancing both basic research and clinical applications.

What are the emerging applications of IMMT antibodies in single-cell and spatial transcriptomics research?

The integration of IMMT antibodies with cutting-edge single-cell and spatial transcriptomics technologies is opening new frontiers in understanding mitochondrial biology in complex tissues and disease states:

Single-Cell Resolution Applications:

• Single-cell protein-RNA correlation:

  • Combining IMMT antibody staining with single-cell RNA sequencing

  • "The Tumor Immune Single Cell Hub (TISCH) was utilized to conduct single-cell analyses to determine which BC cell types may express IMMT"

  • This approach reveals cell type-specific expression patterns and regulatory mechanisms

• CITE-seq and related technologies:

  • Using oligonucleotide-labeled IMMT antibodies for simultaneous protein and transcriptome profiling

  • Enables correlation of IMMT protein levels with global transcriptional states

  • Particularly valuable for identifying regulatory networks controlling IMMT expression

• CyTOF and spectral flow cytometry:

  • Metal-conjugated IMMT antibodies for high-parameter cytometry

  • Flow cytometry applications have been validated using 0.20 μg per 10^6 cells

  • Allows quantification of IMMT alongside numerous other markers to identify distinct cellular phenotypes

Spatial Mapping Technologies:

• Spatial transcriptomics integration:

  • "SpatialDB was used to analyze the spatial transcriptomics, whereby the gene expression in tissue sections can be visualized and quantified"

  • Combining IMMT antibody staining with spatial transcriptomics reveals tissue-specific expression patterns

  • Critical for understanding regional variations in mitochondrial function within heterogeneous tissues

• Multiplexed immunofluorescence/immunohistochemistry:

  • Sequential staining or spectral unmixing enables visualization of IMMT alongside numerous markers

  • Antibody 10179-1-AP has been validated for IHC in human breast cancer tissue at 1:50-1:500 dilution

  • Cyclic immunofluorescence can map IMMT distribution in relation to dozens of cellular structures

• Spatial proteomics approaches:

  • CODEX and similar technologies for highly multiplexed protein mapping

  • In situ proximity ligation assays to visualize IMMT interactions with other proteins

  • These approaches reveal spatial organization of MICOS complexes within tissues

Clinical and Translational Applications:

• Cancer heterogeneity mapping:

  • "IMMT expression in the immune cells of BC tissue was determined based on the GSE114724 dataset"

  • Single-cell approaches reveal tumor-specific and stromal expression patterns

  • Correlation with patient outcomes: "High IMMT expression served as an independent diagnostic biomarker, correlated with advanced clinical status"

• Biomarker development:

  • Integration of spatial and single-cell data for improved prognostic models

  • "The IMMT expression levels demonstrated a high diagnostic accuracy (AUC: 0.701, 95% CI 0.65–0.74)"

  • Potential for developing targeted therapeutic approaches

• Disease mechanism elucidation:

  • Mapping mitochondrial dysfunction at single-cell resolution in complex diseases

  • Understanding cell type-specific vulnerabilities to mitochondrial stress

  • Identifying compensatory mechanisms in cells with altered IMMT expression

Methodological Considerations:

• Antibody validation for new platforms:

  • Verify IMMT antibody performance in fixation conditions compatible with spatial technologies

  • Optimize staining protocols for maximum sensitivity with minimal background

  • Implement appropriate controls specific to multiplexed methods

• Computational analysis integration:

  • Develop analysis pipelines integrating antibody-based and transcriptomic data

  • Apply machine learning approaches to identify patterns across multi-omic datasets

  • Standardize quantification methods across platforms for comparable results

The combination of these emerging technologies with validated IMMT antibodies provides unprecedented insights into mitochondrial biology across diverse biological contexts, from developmental processes to disease mechanisms, with significant implications for both basic research and clinical applications.

How might IMMT antibodies contribute to therapeutic development for diseases with mitochondrial dysfunction?

IMMT antibodies are increasingly valuable tools in therapeutic development for diseases with mitochondrial dysfunction, serving multiple roles from target discovery to treatment monitoring:

Target Identification and Validation:

• Disease association studies:

  • IMMT expression correlates with disease progression in multiple cancers

  • "High IMMT expression is associated with poorer prognosis of patients with lung adenocarcinoma"

  • In breast cancer, "high IMMT expression served as an independent diagnostic biomarker, correlated with advanced clinical status"

• Mechanism elucidation:

  • IMMT antibodies reveal altered mitochondrial architecture in disease states

  • "GSEA identified IMMT perturbation as involved in cell cycle progression and mitochondrial antioxidant defenses"

  • Experimental knockdown studies show that "IMMT impeded the migration and viability of BC cells, arrested the cell cycle, disturbed mitochondrial function, and increased the ROS level"

• Therapeutic target qualification:

  • Immunohistochemistry (1:50-1:500 dilution) can assess IMMT distribution in patient samples

  • Flow cytometry (0.20 μg per 10^6 cells) quantifies expression levels in specific cell populations

  • These applications help determine which patients might benefit from IMMT-targeted therapies

Therapeutic Antibody Development:

• Therapeutic antibody design:

  • Computational approaches enable "the design of antibodies with customized specificity profiles"

  • For IMMT, this could yield antibodies that recognize disease-specific conformations or modifications

  • Engineered antibodies could potentially modulate MICOS complex formation in therapeutic applications

• Antibody-drug conjugates (ADCs):

  • IMMT antibodies could deliver therapeutic payloads to cells with aberrant mitochondrial function

  • Particularly promising for cancers where high IMMT expression correlates with poor prognosis

  • ADCs might selectively target cells dependent on altered mitochondrial dynamics

• Cell-penetrating antibodies:

  • Engineered variants that can access intracellular IMMT

  • Potential to modulate mitochondrial dynamics by interfering with MICOS assembly

  • May stabilize compromised mitochondrial architecture in degenerative conditions

Companion Diagnostics and Therapy Monitoring:

• Patient stratification:

  • IMMT expression and localization patterns could identify responder populations

  • "The specificity of RNA-seq data increased with a higher cut-off value in normal tissue"

  • IHC protocols (1:50-1:500) can assess expression in patient biopsies

• Treatment response monitoring:

  • Serial sampling to track changes in IMMT levels and mitochondrial morphology

  • Flow cytometry applications allow quantitative assessment in blood and bone marrow samples

  • Changes in IMMT expression patterns may serve as early indicators of treatment efficacy

• Resistance mechanism identification:

  • IMMT antibodies can help identify adaptive changes in mitochondrial networks

  • Combined with functional assays to understand metabolic adaptations

  • May guide second-line therapy selection based on mitochondrial phenotype

Emerging Therapeutic Approaches:

• Small molecule discovery:

  • IMMT antibodies enable high-throughput screening for compounds that modulate its function

  • "Pyridostatin acted as a potent drug candidate in BC cells harboring an elevated IMMT expression"

  • Immunoprecipitation applications (0.5-4.0 μg antibody) can identify binding partners for drug targeting

• Gene therapy monitoring:

  • IMMT antibodies can assess the efficacy of genetic interventions targeting mitochondrial dynamics

  • Particularly relevant given that "genetic ablation of Immt induces a lethal disruption of the MICOS complex"

  • Western blotting (1:5000-1:50000) provides quantitative assessment of intervention effects

• Mitochondrial transplantation approaches:

  • IMMT antibodies can verify the integrity of isolated mitochondria for therapeutic transplantation

  • Assess integration of transplanted organelles into recipient cells

  • Track long-term stability and function of engineered mitochondrial networks