Ace Antibody, FITC conjugated

What is the optimal storage condition for ACE Antibody-FITC conjugates?

ACE antibody-FITC conjugates should be stored at -20°C as aliquots to minimize freeze-thaw cycles, which can compromise antibody integrity. For short-term storage (up to one month), the reconstituted antibody can be kept at 4°C. The recommended storage buffer typically contains 0.01M TBS (pH 7.4) or PBS with stabilizing agents such as BSA (1%), preservatives like Proclin-300 (0.03-0.05%), and glycerol (50%) to prevent freeze damage . Light exposure should be minimized as FITC is photosensitive, with excitation/emission wavelengths of 499/515nm . Proper storage is crucial for maintaining consistent signal intensity and minimizing background in flow cytometry applications.

What are the recommended dilutions for different applications of ACE Antibody-FITC?

The optimal dilution varies by application and should be determined experimentally for each lot:

For quantitative experiments, a titration curve should be performed to determine the optimal signal-to-noise ratio for your specific experimental conditions .

How do I properly validate ACE Antibody specificity for my target tissue?

Proper validation requires multiple approaches:

• Include appropriate positive controls where ACE expression is well-established (e.g., lung tissue, vascular endothelium, renal proximal tubules)

• Use negative controls including isotype controls and ACE-null tissues when available

• Confirm reactivity with your species of interest; while some ACE antibodies react with human, mouse, rat, and dog samples, cross-reactivity with other species (e.g., pig, cow, sheep) should be experimentally verified

• Consider complementary techniques (Western blot alongside immunostaining) to confirm specificity

• If discrepancies arise, peptide blocking experiments can help confirm binding specificity to the ACE epitope

Literature indicates ACE expression in multiple tissues including lung, brain, liver, plasma, testis, and umbilical vein endothelial cells that can serve as validation reference points .

How can I optimize intracellular staining of ACE in flow cytometry experiments?

For optimal intracellular ACE staining in flow cytometry:

• Begin with proper fixation using paraformaldehyde (typically 4%) for 10-15 minutes at room temperature

• Use an appropriate permeabilization buffer containing 0.1% Triton X-100 or saponin-based buffers for 15-30 minutes

• Block non-specific binding with 1-5% BSA in permeabilization buffer

• Titrate antibody concentration (typically starting at 1-5 μl per 10^6 cells) to determine optimal signal-to-noise ratio

• Include appropriate compensation controls when multiplexing with other fluorophores, as FITC has potential spectral overlap with PE

• Use a 488nm laser line for FITC excitation, with emission collection around 515nm

• For ACE specifically, be aware that it localizes to both cell membrane and intracellular compartments, so gating strategies should account for this dual distribution

For experiments examining ACE in the endosomal pathway where MHC class II processing occurs, additional permeabilization optimization may be required .

What are the key differences between somatic and germinal forms of ACE that might affect antibody binding?

When selecting an ACE antibody, it's crucial to understand which isoform you're targeting:

• Somatic ACE (sACE) is approximately 170 kDa and contains two homologous domains (N and C domains), each with an active site

• Germinal or testicular ACE (tACE) is approximately 90 kDa and contains only the C-domain

Tissue distribution varies significantly between isoforms:

• sACE: Widely expressed in endothelial cells (especially lung), epithelial cells (proximal renal tubules, intestine), neuronal cells, and some macrophages

• tACE: Primarily expressed in developing male germ cells

For experiments requiring isoform-specific detection, carefully review the immunogen information and epitope location before selection .

How does ACE expression change under inflammatory conditions, and how should this inform experimental design?

ACE expression is dynamically regulated during inflammation, which has important experimental implications:

• IFN-γ and certain pathogens (e.g., L. monocytogenes) induce ACE expression in antigen-presenting cells (APCs)

• This upregulation appears to be physiologically advantageous during immunological challenges

• When designing immunology experiments, consider that:

  • Baseline ACE expression may vary between inflammatory states

  • ACE inhibitors can alter MHC class II antigen presentation efficiency

  • Temporal changes in ACE expression may occur during the inflammatory response

For experiments examining inflammation-related changes:

• Include time-course analyses

• Consider flow cytometry with quantitative beads for accurate expression level assessment

• Include ACE inhibitor controls (e.g., lisinopril at 1 μM) to distinguish enzymatic activity from other protein functions

• Account for potential differences in ACE expression when comparing diseased versus healthy tissues

How does ACE enzymatic activity influence MHC class II antigen presentation, and how can this be experimentally assessed?

ACE plays a significant role in MHC class II antigen presentation through its peptidase activity:

• ACE is present in the endosomal pathway where MHC class II peptide processing and loading occur

• The efficiency of presenting MHC class II epitopes from antigens like ovalbumin (OVA) and hen egg lysozyme (HEL) is markedly affected by cellular ACE levels

• For experimental assessment:

  • Compare antigen presentation using cells from ACE knockout, wild-type, and ACE-overexpressing (ACE10) mice

  • Measure T cell activation (CD69 expression) after 4-hour co-incubation with antigen-presenting cells

  • Quantify cytokine production (e.g., IL-2) in supernatants after 18 hours

  • Use ACE inhibitors (e.g., lisinopril at 1 μM) to distinguish enzymatic from non-enzymatic effects

Experimental setup for measuring ACE's influence on antigen presentation:

• Feed macrophages with antigen (OVA at 100 μg/ml or HEL at 5 mg/ml) for 2 hours

• Fix cells with 1% paraformaldehyde

• Co-incubate with antigen-specific T cells (e.g., OT-II T cells for OVA)

• Measure T cell activation by CD69 expression or cytokine production

This approach has revealed that ACE overexpression enhances MHC class II presentation of certain epitopes, suggesting a novel immunomodulatory role beyond its classical function in the renin-angiotensin system .

What confocal microscopy approaches best reveal ACE's subcellular localization in relation to the antigen processing machinery?

For high-resolution imaging of ACE's subcellular localization:

• Seed macrophages (preferably from ACE10 mice for enhanced signal) onto chamber slides

• Perform phagocytosis assays using 1μm latex beads (1:500 dilution) with 1-hour co-incubation

• Fix cells with 4% PFA/PBS for 10 minutes

• Block and permeabilize with 1% BSA and 0.1% Triton X-100 in PBS for 1 hour

• For co-localization studies, perform double immunostaining:

  • Primary ACE antibody (rabbit polyclonal) detected with Alexa Fluor 488-conjugated secondary antibody

  • Primary antibodies against endosomal/lysosomal markers detected with Alexa Fluor 594-conjugated secondary antibodies

• Mount with DAPI-containing medium for nuclear visualization

• Image using confocal microscopy with appropriate laser settings (488nm for FITC)

For tracking ACE through the endocytic pathway, stain for these markers:

• Early endosomes: EEA1

• Late endosomes: Rab7

• Lysosomes: LAMP1

• MHC class II compartments: HLA-DM or MIIC markers

This approach has revealed that ACE is present in the endosomal pathway, positioning it to influence peptide processing for MHC class II presentation .

How can ACE antibodies be used to investigate the relationship between ACE expression levels and immune response magnitude in vivo?

To investigate how ACE expression affects immune responses in vivo:

• Compare immune responses in wild-type versus ACE-overexpressing (ACE10) mice:

  • Immunize mice with antigen (e.g., 100 μg OVA emulsified in CFA) subcutaneously

  • After 9 days, collect spleens and draining lymph nodes

  • Restimulate cells with specific peptides (e.g., OVA 323-339)

  • Measure T cell responses via cytokine production (IFN-γ, IL-17A) by ELISA

  • Assess antibody production against the immunizing antigen

• For more detailed analysis:

  • Flow cytometry using ACE-FITC antibodies can quantify ACE expression on different APC populations

  • Correlation analyses between ACE expression levels and immune response magnitude

  • Adoptive transfer experiments with ACE-high versus ACE-low APCs

Research has shown that ACE10 mice (over-expressing ACE in myeloid cells) generate substantially stronger CD4+ T cell responses (5.52-fold higher IFN-γ and 5.48-fold higher IL-17) and antibody responses (>20-fold higher IgG1) compared to wild-type mice when immunized with ovalbumin . This suggests ACE expression levels can be manipulated to enhance vaccine responses or modulate autoimmunity.

How can background fluorescence be minimized when using ACE Antibody-FITC in tissues with high autofluorescence?

When working with tissues that exhibit high autofluorescence (particularly lung, where ACE is highly expressed):

• Perform autofluorescence quenching:

  • Treat sections with 0.1-1% Sudan Black B in 70% ethanol for 20 minutes

  • Alternatively, use commercial autofluorescence quenching reagents

  • Consider specialized quenching protocols for formalin-fixed tissues (e.g., sodium borohydride treatment)

• Optimize antibody dilution:

  • Titrate antibody concentrations more carefully for autofluorescent tissues

  • Generally, higher dilutions (1:100-1:200) may help reduce background

• Imaging considerations:

  • Use narrow bandpass filters to minimize capture of autofluorescence

  • Consider spectral unmixing during confocal microscopy

  • Capture autofluorescence in an empty channel for digital subtraction

• Controls are essential:

  • Include unstained tissue sections

  • Use isotype-FITC controls at the same concentration

  • Consider alternative fluorophores (e.g., Alexa Fluor 647) for highly autofluorescent tissues

What are the critical factors to consider when using ACE Antibody-FITC to study endothelial cells in different vascular beds?

Endothelial cells represent a primary site of ACE expression, but significant heterogeneity exists across vascular beds:

• Expression level variations:

  • Pulmonary endothelial cells express the highest levels of ACE

  • Brain endothelial cells (blood-brain barrier) have moderate expression

  • Hepatic sinusoidal endothelial cells have lower expression

• Protocol adjustments:

  • Titrate antibody concentration for each vascular bed

  • For flow cytometry of primary endothelial cells, use 1-5 μl per 10^6 cells as starting point

  • Include endothelial markers (CD31, VE-cadherin) in multi-parameter analyses

• Isolation considerations:

  • Fresh isolation is preferable as ACE can be sensitive to enzymatic digestion methods

  • When using collagenase for endothelial isolation, optimize concentration and duration

  • For human umbilical vein endothelial cells, ACE can be detected and has been studied extensively

• Physiological vs. pathological conditions:

  • ACE expression in endothelial cells changes with inflammation, hypoxia, and other stressors

  • Include proper temporal controls when studying dynamic processes

How can ACE antibodies be used to investigate the differential effects of ACE inhibitors versus angiotensin receptor blockers on immune function?

This advanced application requires careful experimental design:

• In vitro assessment of drug effects on ACE-dependent antigen presentation:

  • Treat antigen-presenting cells with ACE inhibitors (e.g., lisinopril at 1 μM) or angiotensin receptor blockers

  • Measure antigen presentation efficiency using T cell activation assays

  • Use ACE-FITC antibody to quantify surface vs. intracellular ACE with flow cytometry

• For mechanistic investigations:

  • ACE-FITC antibody can distinguish between drug effects on expression versus activity

  • Use enzyme activity assays alongside ACE-FITC staining to correlate protein levels with function

  • Investigate potential conformational changes induced by inhibitors using competitive binding assays

• In vivo comparative studies:

  • Compare immune responses in animals treated with ACE inhibitors versus angiotensin receptor blockers

  • Use flow cytometry with ACE-FITC antibody to track changes in ACE expression on immune cell populations

  • Correlate with functional immune readouts (e.g., T cell responses, antibody production)

Research indicates that ACE inhibitors may affect immune function through mechanisms beyond angiotensin II production blockade, including direct effects on MHC class II peptide processing . This application represents an exciting frontier for understanding the immunomodulatory effects of these widely used cardiovascular drugs.

How can ACE antibodies contribute to understanding the role of the renin-angiotensin system in COVID-19 pathophysiology?

Given that SARS-CoV-2 uses ACE2 (distinct from ACE) for cell entry, ACE antibodies enable important comparative studies:

• Dual staining approaches:

  • Use ACE-FITC antibody alongside ACE2 antibodies (different fluorophore) to track expression changes

  • Monitor relative expression levels in infected versus uninfected tissues

  • Investigate potential compensatory regulation between ACE and ACE2

• Immune cell infiltration studies:

  • Track ACE expression on infiltrating macrophages in COVID-19 lung tissues

  • Correlate with disease severity and inflammatory markers

  • Investigate whether ACE expression affects antigen presentation of viral peptides

• Therapeutic implications:

  • Use ACE-FITC antibodies to monitor effects of ACE inhibitors on immune cell function

  • Investigate whether altered ACE expression affects COVID-19 vaccine responses

  • Study how COVID-19 infection alters the balance of the renin-angiotensin system components

Since ACE plays a role in MHC class II antigen presentation , investigating its expression during COVID-19 may provide insights into abnormal immune responses observed in severe disease.