ESR2 Antibody

Which ESR2 antibodies have been rigorously validated for immunohistochemistry?

The issue of antibody validation highlights how inadequate validation has potentially misled ESR2 research for years, with many studies potentially overestimating ESR2 expression patterns due to non-specific antibody binding .

How can I determine if my ESR2 antibody is specific?

Methodological approach to validating ESR2 antibody specificity:

• Use multiple validation techniques concurrently:

  • Western blot analysis with appropriate positive and negative controls

  • Immunohistochemistry on known positive tissue (e.g., granulosa cells in ovaries) and negative controls

  • Testing in cell lines with confirmed ESR2 expression by RNA-seq or qPCR

  • Ideally, verification by immunoprecipitation followed by mass spectrometry

• Essential controls include:

  • Positive control: Cell lines transfected with ESR2 expression vectors

  • Negative control: ESR2 knockout models or cell lines with confirmed absence of ESR2 expression

  • Testing on tissues with verified ESR2 mRNA expression using RNA-seq

Many published studies used inadequate validation, relying only on expected molecular weight bands rather than comprehensive specificity testing, which has contributed to contradicting results in the field .

What are the differences between commonly available ESR2 antibodies?

The table below summarizes key characteristics of several ESR2 antibodies:

Sources:

What are the optimal conditions for detecting ESR2 using immunohistochemistry?

Optimized protocol for ESR2 immunohistochemistry varies by species and tissue type:

For mouse tissues:

• Heat-induced antigen retrieval is critical (citrate buffer pH 6.0 or TE buffer pH 9.0)

• When using PPZ0506: dilution of 1:2000 in PBST

• Mouse-on-mouse polymer IHC kit is recommended to reduce background

• Visualization with SG substrate provides optimal signal-to-noise ratio

• Critical blocking step: use Mouse-on-Mouse Polymer IHC Kit with horseradish peroxidase polymer detector reagent (15 min at room temperature)

For rat tissues:

• Antigen retrieval is essential but may require optimization by tissue

• When using PPZ0506: dilution of 1:2000 in 5% normal goat serum in PBST

• Incubation with 50% goat anti-mouse IgG-HRP labeled polymer for 2 hours at room temperature

• Visualization with ImmPACT SG substrate kit

The critical difference between successful and unsuccessful staining often lies in the intensity of antigen retrieval and the appropriate blocking steps to reduce non-specific binding .

How should Western blot protocols be optimized for ESR2 detection?

Optimized Western blot protocol for ESR2:

• Sample preparation:

  • Include positive controls (e.g., MCF-7 cells, mouse testis tissue)

  • Include negative controls (verified by RNA-seq)

  • Load 30μg of protein per lane under reducing conditions

• Gel electrophoresis:

  • 5-20% SDS-PAGE gradient gel

  • Run at 70V (stacking)/90V (resolving) for 2-3 hours

• Transfer conditions:

  • Transfer to nitrocellulose membrane at 150mA for 50-90 minutes

• Blocking:

  • 5% non-fat milk in TBS for 1.5 hours at room temperature

• Primary antibody:

  • PPZ0506 (1:500 to 1:2000) or other validated antibody

  • Incubate overnight at 4°C

• Washing:

  • TBS with 0.1% Tween, 3 times for 5 minutes each

• Secondary antibody:

  • Anti-mouse or anti-rabbit IgG-HRP (depending on primary)

  • Dilution 1:500 to 1:1000

  • Incubate for 1.5 hours at room temperature

• Detection:

  • Enhanced chemiluminescence

  • Expected molecular weight for ESR2: 59-60 kDa (though observed weight may range from 50-60 kDa)

The most critical steps for successful ESR2 Western blot are using adequately validated antibodies and including proper positive and negative controls to confirm specificity .

What are the recommended approaches for resolving contradictory ESR2 expression data?

To resolve contradictory ESR2 expression data, a multi-platform approach is recommended:

• Validate antibody specificity first:

  • Test on positive and negative control tissues

  • Confirm with knockout/knockdown models if available

  • Compare results with mRNA expression data

• Employ multiple detection methods:

  • Compare protein detection (IHC, WB) with mRNA detection (qPCR, RNA-seq)

  • Use Parallel Reaction Monitoring (PRM) for targeted proteomics approach as a non-antibody dependent method

  • Consider Rapid Immunoprecipitation Mass spectrometry of Endogenous protein (RIME) to confirm protein identity

• Address species differences systematically:

  • Document species-specific expression patterns separately

  • Note that ESR2 expression patterns differ between humans, mice, and rats

  • Avoid generalizing findings across species without validation

• Consider methodological variables:

  • Different fixation methods affect epitope availability

  • Antigen retrieval conditions significantly impact results

  • Tissue processing methods should be standardized

Research by Andersson et al. demonstrated how different antibodies gave contradictory results even in the same tissues, highlighting the importance of rigorous antibody validation and multi-method confirmation of findings .

What is the actual tissue distribution of ESR2 protein based on validated antibody studies?

Accurate ESR2 protein distribution based on studies using the validated antibody PPZ0506:

In humans:

• Testis: Moderate to high expression

• Ovary: Moderate expression in granulosa cells

• Placenta: Weak expression

• Lymphoid cells: Present in specific subpopulations

• Limited or absent in most other tissues including breast tissue

• Tumor expression: Found in granulosa cell tumors, subset of malignant melanoma and thyroid cancers

In mice:

• Ovary: Strong expression in granulosa cells

• Testis: Specific cell populations show expression

• Brain: Limited to specific nuclei

• Minimal to absent expression in most peripheral tissues

In rats:

• More localized distribution than previously assumed

• Expression pattern differs from mice and humans in some tissues

• Specific brain regions show nuclear localization

This restricted expression pattern contradicts many earlier studies that suggested widespread ESR2 expression across multiple tissues. The discrepancy is attributed to non-specific binding of inadequately validated antibodies .

What are the sex and species differences in ESR2 expression patterns?

Key findings on sex and species differences in ESR2 expression:

Sex differences:

• In brain regions, many nuclei show sexually dimorphic expression patterns

• Estrogen treatment affects ESR2 expression differently in males versus females

• In the diestrus phase, female mice and rats show distinct ESR2 distribution in hypothalamic and other brain regions compared to males

Species differences:

• Mouse, rat, and human ESR2 expression patterns show notable differences even in homologous tissues

• In peripheral tissues, rats show distinctive expression patterns compared to mice

• Brain expression of ESR2 shows both conservation and divergence across rodent species

Studies using the validated antibody PPZ0506 revealed that previous assumptions about conserved expression across species were often inaccurate. This has significant implications for translating findings from animal models to human applications .

How does ESR2 expression correlate with ESR1 (ERα) expression in different tissues and cancers?

The relationship between ESR2 and ESR1 expression varies by tissue and disease state:

• Normal tissues:

  • Generally show inverse correlation patterns

  • ESR1 dominates in reproductive tissues like uterus and mammary gland

  • ESR2 is more prominent in ovary, parts of the brain, and immune cells

• Breast cancer:

  • RNA-seq data from large cohorts show a weak inverse correlation (Spearman R = -0.20)

  • ESR2 mRNA is expressed at much lower levels than ESR1

  • ESR2 is relatively higher in ERα-negative subtypes (normal-like, basal-like, and HER2-enriched)

  • Lower ESR2 expression observed in ERα-positive subtypes (luminal A and B)

• Expression by molecular subtype:

  • Basal-like breast cancers: Higher ESR2, lower ESR1

  • Luminal A/B cancers: Higher ESR1, lower ESR2

  • Age-related patterns: ESR1 expression increases with patient age, while ESR2 remains relatively stable

How can ESR2 antibodies be applied in cell-type specific colocalization studies?

Methodology for ESR2 colocalization studies:

• Double immunohistochemistry protocol:

  • First detect ESR2 using optimized protocols with PPZ0506

  • Remove antibodies using glycine-HCl buffer (pH 2.2, 90 min at room temperature)

  • Proceed with second marker detection (e.g., oxytocin or arginine vasopressin)

  • Alternative approach: fluorescent double-labeling with compatible secondary antibodies

• Applications in neuroscience:

  • ESR2+/OXT+ and ESR2+/AVP+ cell populations can be quantified

  • Regional distribution in hypothalamic and other brain nuclei can be mapped

  • Sexual dimorphism in colocalization patterns can be assessed

• Cell counting methodology:

  • Define regions of interest using Nissl staining to identify neural nuclei

  • Count cell nuclei with immunopositive signals manually or with assisted software

  • Include blinding procedures to eliminate observer bias

  • Express results as density (cells/mm²) or percentage of total cells

This approach has revealed specific cell populations that express ESR2 in conjunction with other markers, providing insights into the functional significance of ESR2 in different cellular contexts .

What is the significance of ESR2 in cancer research based on validated antibody studies?

The significance of ESR2 in cancer research has been reassessed based on studies using validated antibodies:

These findings highlight how inadequately validated antibodies led to overestimation of ESR2's potential as a therapeutic target in diverse cancers .

How should researchers integrate ESR2 mRNA and protein expression data?

Methodological approach to integrating ESR2 mRNA and protein data:

• Recognize the strengths and limitations of each approach:

  • RNA-seq provides quantitative expression levels but doesn't confirm protein translation

  • qPCR offers high sensitivity for mRNA but similar limitations for protein inference

  • Validated antibody IHC provides spatial information and confirms protein presence

  • Western blot confirms size but lacks spatial information

• Best practices for integration:

  • First establish baseline using RNA-seq or qPCR to identify tissues with detectable transcripts

  • Follow with validated antibody-based methods in tissues with detectable transcripts

  • Document discrepancies between mRNA and protein detection

  • Consider post-transcriptional regulation when differences are observed

• Approach to contradictory findings:

  • When protein is detected without corresponding mRNA, question antibody specificity

  • When mRNA is detected without corresponding protein, investigate post-transcriptional mechanisms

  • Consider sensitivity thresholds of each method

  • Evaluate tissue/cell heterogeneity as potential explanation

What are the most common artifacts in ESR2 immunohistochemistry and how can they be avoided?

Common artifacts and solutions in ESR2 immunohistochemistry:

• Non-specific nuclear staining:

  • Cause: Inadequate blocking, particularly in mouse tissues using mouse antibodies

  • Solution: Use Mouse-on-Mouse Polymer IHC Kit; increase blocking time; optimize antibody dilution (1:2000 recommended for PPZ0506)

• Cytoplasmic background:

  • Cause: Insufficient washing; non-specific binding

  • Solution: Increase wash duration and frequency; add 0.1% Tween to PBS; use 5% normal goat serum in blocking buffer

• Loss of antigenicity:

  • Cause: Inadequate antigen retrieval; overfixation

  • Solution: Optimize antigen retrieval (intense heat-induced retrieval required); test both citrate buffer pH 6.0 and TE buffer pH 9.0; standardize fixation time

• False positives in expected ESR2-negative tissues:

  • Cause: Non-specific antibody binding; endogenous peroxidase activity

  • Solution: Include validated negative controls; block endogenous peroxidase with hydrogen peroxide treatment; validate antibody specificity

• Variability between replicates:

  • Cause: Inconsistent processing; antibody batch variation

  • Solution: Standardize all protocol steps; process control and experimental samples simultaneously; maintain consistent antibody lots when possible

The development of optimized protocols for mouse and rat tissues has significantly reduced artifacts and improved reproducibility in ESR2 immunohistochemistry .

What storage and handling conditions are optimal for maintaining ESR2 antibody performance?

Optimal storage and handling conditions for ESR2 antibodies:

• Long-term storage:

  • Store at -20°C for maximum stability

  • Antibodies are typically stable for one year after shipment when stored properly

  • For some antibodies like PPZ0506, aliquoting is unnecessary for -20°C storage

• Working stock handling:

  • For frequent use, store small aliquots at 4°C for up to one month

  • Avoid repeated freeze-thaw cycles which degrade antibody performance

  • Return to -20°C promptly after use

• Buffer considerations:

  • Most ESR2 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

  • Some preparations include 0.1% BSA for additional stability

  • Do not dilute stock antibody unless preparing working solution

• Shipping and temporary storage:

  • Typically shipped on blue ice

  • Can withstand room temperature for short periods during shipping

  • Upon receipt, immediately transfer to recommended storage conditions

• Antibody dilutions:

  • Prepare fresh working dilutions on the day of experiment

  • Use recommended diluent (typically PBS with 0.1% Tween or 5% normal serum)

  • Discard unused diluted antibody after experiment

Following these storage and handling practices helps maintain antibody specificity and sensitivity, particularly important for ESR2 antibodies given the challenges in this field .

How can researchers quantitatively assess ESR2 expression in immunohistochemistry studies?

Methodology for quantitative assessment of ESR2 expression in IHC:

• Cell counting approach:

  • Define regions of interest using anatomical landmarks or Nissl staining

  • Count ESR2-immunopositive nuclei manually or using digital image analysis

  • Express as density (cells/mm²) or percentage of total cells

  • Include blinding procedures to eliminate observer bias

• Digital image analysis:

  • Capture images under standardized conditions (same microscope, settings, and exposure)

  • Use software like ImageJ to trace regions of interest

  • Apply threshold to identify positive nuclei

  • Measure parameters such as staining intensity, percentage of positive cells, and H-score

• Semi-quantitative scoring:

  • Score intensity (0 = negative, 1 = weak, 2 = moderate, 3 = strong)

  • Assess percentage of positive cells (0-100%)

  • Calculate H-score = Σ(intensity × percentage), ranging from 0-300

  • Require multiple independent observers for reliability

• Considerations for accuracy:

  • Include positive and negative controls in each staining batch

  • Normalize results using internal reference standards

  • Account for tissue heterogeneity by analyzing multiple fields

  • Document staining conditions precisely for reproducibility

Studies examining sex differences and effects of estrogen manipulation on ESR2 expression have successfully used these quantitative approaches to detect biologically meaningful differences in expression patterns .