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ESR2 / Estrogen receptor beta · IHC design guide
Design Immunohistochemistry for ESR2
ESR2 shows low nuclear staining in several tissues, including testis and ovary (HPA tissue IHC). Use this guide to plan fixation, controls, and nuclear scoring for chromogenic IHC on paraffin sections.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ESR2 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Nuclear staining in tissue sections (HPA tissue IHC) | |
| Staining pattern | Low nuclear staining in Leydig and ovarian stromal cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adrenal gland | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Weak staining and medium antibody–RNA consistency (HPA tissue IHC) | |
| Regulation | Expression varies by tissue (UniProt) | |
| Isoform / epitope | 9 isoforms; verify epitope coverage (UniProt) |
Section 1
Recommended ESR2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with published ESR2 IHC methods from PMC9631985, PMC10726529, and PMC9141030.
| Sample | Paraffin-embedded human cervix cancer tissue; fixative not specified (datasheet M00786-1) |
| Fixation | Image fixative and duration unreported (datasheet M00786-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit monoclonal (clone IDI-5) anti-ESR2, 1:50 (datasheet M00786-1) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ESR2-positive staining in cells in zona glomerulosa of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Low nuclear expression in several tissues, including testis and ovaries. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min (page retrieval setting); use citrate pH 6.0 when reproducing the cited brain or ovary methods (PMC9631985; PMC10726529).
Section 2
What Is the Expected ESR2 Staining Pattern?
ESR2 is a nuclear receptor with no transmembrane segment (UniProt Q92731: subcellular location and topology). In paraffin-section IHC, expect nuclear staining in selected cells: HPA reports medium staining in adrenal zona glomerulosa cells and low staining in testis Leydig cells and ovarian stroma cells (HPA tissue IHC). HPA labels its tissue IHC reliability Enhanced while noting medium consistency between antibody staining and RNA expression (HPA tissue IHC).
| Distinct nuclear signal in adrenal zona glomerulosa cells. | This matches HPA’s medium staining in that cell population and ESR2’s nuclear location (HPA tissue IHC; UniProt Q92731). Judge localization and cell identity together; intensity alone does not establish specificity (general IHC practice). |
| Weak nuclear signal in testis Leydig cells or ovarian stroma cells. | Low staining is consistent with the reported pattern (HPA tissue IHC). A weak result in either population should be interpreted against a positive control and local background, since HPA’s tissue profile does not promise strong staining in every section (general IHC practice; HPA tissue IHC). |
| Predominantly cytoplasmic or membrane staining, with little nuclear signal. | That is discordant with the expected IHC compartment (UniProt Q92731: nucleus; HPA tissue IHC: low nuclear expression). Review background and antibody specificity before calling the cells ESR2-positive (general IHC practice). HPA’s uncertain vesicle observation comes from ICC-IF and does not validate cytoplasmic IHC staining (HPA subcellular ICC-IF). |
| Strong signal in a cell population HPA lists as not detected, such as breast adipocytes. | Treat the result as a specificity or detection-control concern, not proof that the whole tissue lacks ESR2 (HPA tissue IHC: breast adipocytes not detected; general IHC practice). Check cell identification, cross-reactivity and endogenous chromogenic activity (general IHC practice). |
| Diffuse colour across nuclei, cytoplasm and surrounding tissue. | The spread obscures ESR2’s expected nuclear pattern (UniProt Q92731; HPA tissue IHC). Assess nonspecific antibody binding, incomplete blocking and detection background with an appropriate negative control before scoring positive nuclei (general IHC practice). |
💡Expected ESR2 appearanceCall a section positive when identifiable adrenal zona glomerulosa cells show predominantly nuclear, medium-intensity staining, with suitably low nuclear staining also plausible in Leydig or ovarian stroma cells; broad cytoplasmic colour or strong staining in an HPA-negative cell population is suspect (HPA tissue IHC; UniProt Q92731).
| Cell population and expected intensity | HPA reports medium staining in adrenal zona glomerulosa cells but low staining in Leydig and ovarian stroma cells (HPA tissue IHC). Select and score controls by the named cell population; a tissue name alone is insufficient (general IHC practice). |
| Subcellular location and protein topology | ESR2 is nuclear and has no transmembrane segment or signal peptide (UniProt Q92731). A membrane-dominant IHC pattern therefore needs investigation; these annotations do not establish how fixation or antigen retrieval affects the antibody (UniProt Q92731; source-scope limitation). |
| Isoforms and epitope coverage | UniProt lists nine ESR2 isoforms (UniProt Q92731). The payload gives no catalog antibody epitope, so isoform recognition and any resulting tissue-staining differences cannot be assigned from this record (source-scope limitation). |
| IHC evidence and antibody choice | HPA labels the overall tissue IHC reliability Enhanced, while listing CAB079300 as IHC Supported and HPA068406 only as ICC Supported (HPA tissue IHC; HPA antibodies). Apply an IHC-validated antibody to paraffin sections; do not transfer an ICC validation label to IHC (general IHC practice). |
| IF/ICC Q: Where should ESR2 fluorescence appear? | A: Mainly in the nucleoplasm; HPA also reports vesicles with uncertain support (HPA subcellular ICC-IF). That observation answers localization for IF/ICC; it does not supply a paraffin IHC protocol or validate vesicular chromogenic staining (HPA subcellular ICC-IF; source-scope limitation). |
| Situation | Likely cause | Next action |
|---|---|---|
| No nuclear signal in adrenal zona glomerulosa cells. | This misses HPA’s reported medium-positive cell population (HPA tissue IHC); the immediate cause is undetermined. | Confirm the cells and positive control, then check the catalog antibody’s IHC-P instructions for retrieval, dilution and detection conditions (general IHC practice). No ESR2-specific fixation sensitivity is reported in these payloads (source-scope limitation). |
| No signal in Leydig or ovarian stroma cells, while a positive control stains. | These are low-staining populations in HPA’s IHC profile, so a negative read can reflect limited visibility at the assay’s threshold (HPA tissue IHC; general IHC practice). | Review nuclear signal against adjacent background and assess additional sections or the HPA medium-positive adrenal control where available (general IHC practice; HPA tissue IHC). Do not infer complete tissue absence from one negative cell-level read (general IHC practice). |
| Colour is concentrated at cell membranes or throughout cytoplasm. | That distribution conflicts with UniProt’s nuclear annotation and HPA’s nuclear tissue profile (UniProt Q92731; HPA tissue IHC). | Check morphology and the negative control, then reassess antibody specificity and detection background before scoring (general IHC practice). Do not use HPA’s uncertain ICC-IF vesicle finding to justify a membrane IHC call (HPA subcellular ICC-IF). |
| Strong staining appears in breast adipocytes or bone-marrow hematopoietic cells. | HPA lists those particular cell populations as not detected; this discrepancy can reflect cell misidentification, cross-reactivity or detection background (HPA tissue IHC; general IHC practice). | Verify the stained cell type and compare with a no-primary control and a known-positive cell population (general IHC practice). Keep the conclusion limited to the examined cells, since HPA’s negative labels are cell-specific (HPA tissue IHC). |
| Diffuse brown background obscures nuclear boundaries. | Nonspecific binding or endogenous chromogenic activity can make nuclear scoring unreliable (general IHC practice). | Inspect no-primary and detection controls, then review blocking, wash and chromogen steps under the assay’s instructions (general IHC practice). Score ESR2 only where discrete nuclear staining remains distinguishable (UniProt Q92731; general IHC practice). |
| ICC-IF images show vesicular signal, but the paraffin IHC slide shows cytoplasmic colour. | HPA calls the vesicle location uncertain in ICC-IF and reports mainly nucleoplasmic localization; the two assay observations are not interchangeable (HPA subcellular ICC-IF). | Interpret the paraffin slide against the nuclear IHC pattern and its controls (HPA tissue IHC; general IHC practice). Use the separate IF/ICC guide for fluorescence assay design; this IHC result alone cannot resolve the uncertain vesicle annotation (HPA subcellular ICC-IF). |
Experiment evidence
IHC & IF images showing ESR2 staining
M00786-1 · IHCImmunohistochemical analysis of paraffin-embedded human cervix cancer, using Estrogen Receptor beta Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000140009-ESR2/tissue/cerebral+cortex#imid_20558243
HPA · Adrenal glandHuman Protein Atlas IHC, Adrenal gland. Source: https://www.proteinatlas.org/ENSG00000140009-ESR2/tissue/adrenal+gland#imid_1248
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000140009-ESR2/tissue/colon#imid_20555785
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000140009-ESR2/tissue/liver#imid_20555794
HPA · ICC-IF RT-4Human Protein Atlas immunofluorescence, RT-4, antibody HPA068406; green = ESR2, blue = nucleus, red = microtubules.
HPA · ICC-IF RT-4Human Protein Atlas immunofluorescence, RT-4, antibody HPA068406; green = ESR2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ESR2 IHC & IF
🧪Run adrenal gland first: cells in the zona glomerulosa should show nuclear staining (HPA: Medium in cells in the zona glomerulosa; UniProt Q92731: nucleus). Use appendix glandular cells as a negative comparator (HPA: Not detected); other cells on the adrenal section should show only background and should not be assumed negative without validation.
Positive control tissue: Adrenal gland (Cells in zona glomerulosa, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ESR2 in RT-4, SK-MEL-30, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; use host species and isotype-matched nonimmune immunoglobulin for a monoclonal primary, or host-matched nonimmune immunoglobulin for a polyclonal primary; and compare with ESR2 knockout material processed identically (standard IHC controls). Quench endogenous peroxidase in the adrenal section and block endogenous biotin if using avidin–biotin detection (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window and antigen-retrieval dependency are unreported in the supplied evidence; optimise retrieval empirically for paraffin IHC (supplied target/application evidence). The M00786-1 paraffin-section caption does not report a fixative, and the evidence does not establish whether frozen sections or IF would be easier (selected-SKU tissue-IHC caption; supplied target/application evidence). Assess adrenal background with the no-primary control before scoring nuclear signal, since endogenous peroxidase can produce chromogenic background (standard IHC practice; UniProt Q92731: nucleus).
HPA tissue IHC evidence for ESR2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Cells in zona glomerulosa | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ESR2 IHC Tips
Troubleshoot ESR2 staining by checking nuclear signal, cell identity, epitope coverage and matched controls before interpreting intensity.
How should I adjust retrieval when ESR2 nuclear staining is weak?
Start with Tris-EDTA pH 9.0 heat-induced retrieval at 95–98 °C for 20 min (page method: nuclear-antigen rule). Run a known positive section alongside the test section, then compare nuclear signal and tissue preservation at the same antibody concentration (standard IHC practice). If staining remains weak, test a shorter or longer heating interval on adjacent sections while keeping the buffer and detection system constant (standard IHC practice). If that fails, compare an alternative retrieval buffer as a documented fallback, recording its pH and heating time (standard IHC practice). Evaluate improvement in nuclei, because ESR2 is nuclear and HPA reports mainly nucleoplasmic localisation (UniProt Q92731 localisation; HPA subcellular).
Could fixation explain weak or uneven ESR2 staining in paraffin sections?
ESR2-specific fixation sensitivity is unknown from the supplied evidence; the selected M00786-1 caption identifies paraffin sections but does not state a fixative (selected product caption). Record the fixative, fixation duration and processing history for each specimen before comparing staining (standard IHC practice). Compare adjacent sections processed together and stain them in one run, using a nuclear positive control to distinguish specimen variability from a run failure (standard IHC practice; UniProt Q92731 localisation). If staining varies across a section, inspect morphology and retrieval uniformity before changing antibody concentration (standard IHC practice). Do not assign the variation to an ESR2-specific fixation effect without matched evidence.
How should I assess ESR2 staining outside the nucleus?
Prioritise nuclear staining when assessing chromogenic ESR2 IHC, because UniProt places ESR2 in the nucleus and HPA supports nucleoplasmic localisation (UniProt Q92731 localisation; HPA subcellular). HPA also lists vesicular localisation as uncertain, so cytoplasmic puncta alone provide weaker evidence than a reproducible nuclear pattern (HPA subcellular). Compare nuclear and cytoplasmic signal separately in matched positive and negative sections, keeping retrieval and DAB development time constant (standard IHC practice). Review a counterstained section at high magnification to confirm that the brown signal overlaps nuclei rather than adjacent pigment or debris (standard IHC practice). Record any convincing extranuclear pattern separately instead of folding it into a nuclear score (standard IHC practice).
Why might two ESR2 antibodies stain different nuclear populations?
ESR2 has 9 listed isoforms, so antibodies recognising different epitopes may detect different subsets of ESR2 protein (UniProt Q92731 isoforms; standard immunohistochemistry principle). Check each antibody’s documented immunogen or epitope against the isoforms it is intended to detect before comparing staining (standard IHC practice). The ligand-binding domain spans residues 264–498, while reported phosphorylation sites include Ser87 and Ser105; epitope placement can therefore matter when evaluating discrepancies (UniProt Q92731 domains and modified residues). Do not infer an isoform from staining intensity or nuclear location alone (standard IHC practice). If epitope coverage is unspecified, report the result as antibody-dependent ESR2 immunoreactivity and seek orthogonal validation (standard IHC practice).
How can I check an ESR2 IHC pattern with multiplex IF?
For the separate IF/ICC workflow, pair ESR2 with a marker that identifies the expected cell population and score signal within those marker-defined cells (standard IF practice). For example, HPA reports ESR2 staining in ovarian stromal cells and testicular Leydig cells, so choose a validated marker appropriate to the population being examined (HPA tissue IHC; standard IF practice). Select fluorophores away from the tissue’s strongest autofluorescence and include single-stain controls to check spectral spillover (standard IF practice). Because ESR2 has no transmembrane segment and is mainly nucleoplasmic, use a validated mild permeabilisation step to give antibody access to the nuclear epitope (UniProt Q92731 topology; HPA subcellular; standard IF practice). Confirm that the resulting signal overlaps nuclei before comparing it with chromogenic IHC (HPA subcellular; standard IF practice).
What should I check when ESR2 DAB staining looks diffuse?
Compare the test section with a no-primary control processed through the same peroxidase block and DAB development (standard chromogenic IHC practice). If both show brown deposits, inspect endogenous peroxidase activity, pigment and nonspecific secondary-reagent binding before adjusting the ESR2 antibody (standard IHC practice). If background appears only with primary antibody, titrate antibody concentration and blocking conditions on adjacent sections while preserving a detectable nuclear positive control (standard IHC practice; UniProt Q92731 localisation). Examine staining at section edges and damaged areas separately, because these can accumulate misleading signal (standard IHC practice). HPA describes ESR2 tissue expression as generally low and nuclear, so diffuse staining throughout every compartment needs careful validation (HPA tissue IHC).
How should I score heterogeneous ESR2 IHC staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and nuclear positivity threshold before scoring, then keep illumination and DAB development comparable across sections (standard IHC practice). Report the percentage of positive nuclei and an H-score based on nuclear intensity categories 0–3, or report positive nuclei per mm² when cell density varies (standard IHC practice). Normalise positive counts to the number of evaluable nuclei in the specified cell population, excluding folds, necrosis and poorly preserved regions (standard IHC practice). Score cytoplasmic signal separately, because the supported ESR2 location is nucleoplasmic (HPA subcellular). State the antibody, retrieval method, threshold and sampled regions so another reader can assess the comparison (standard IHC practice).
When is an apparent ESR2-positive IHC result convincing?
Look for reproducible nuclear staining in the expected cells across well-preserved regions, with appropriate positive and no-primary controls (UniProt Q92731 localisation; standard IHC practice). HPA reports medium staining in adrenal zona glomerulosa cells and low staining in testicular Leydig cells and ovarian stromal cells, providing cell-specific context rather than a universal intensity cutoff (HPA tissue IHC). Treat staining confined to section edges, necrotic areas or compartments outside the nucleus as provisional until controls resolve it (HPA subcellular; standard IHC practice). Check whether a brown deposit persists without primary antibody, which can indicate endogenous enzyme activity or other background (standard IHC practice). Interpret the result as antibody-dependent ESR2 immunoreactivity when isoform coverage has not been established (UniProt Q92731 isoforms; standard IHC practice).
Boster reagents
Best ESR2 / Estrogen receptor beta IHC Antibodies
An anti-ESR2 antibody has IHC data from paraffin-embedded human cervix cancer (M00786-1 IHC image caption). Human is the only listed species, and no IF image is supplied (catalog reactivity; IF images).
Real IHC data
Anti-Estrogen Receptor beta ESR2 Rabbit Monoclonal Antibody
Cat # M00786-1
M00786-1 is listed for human IHC (catalog applications; reactivity). Its IHC image shows paraffin-embedded human cervix cancer; no IF image is supplied (M00786-1 IHC image caption; IF images).
Which to pick: Choose M00786-1 for human tissue IHC: it is rabbit monoclonal clone IDI-5, and its own image shows paraffin-embedded human cervix cancer (catalog host and clone; M00786-1 IHC image caption). No listed SKU supports an IF/ICC or cross-species recommendation (catalog applications: IHC, WB; reactivity: Human; IF images: none). The caption does not report the fixative (M00786-1 IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q92731 (ESR2_HUMAN, Estrogen receptor beta).
- Human Protein Atlas. ESR2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ESR2 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to vesicles..
- Human Protein Atlas. ESR2 antibody validation summary (2 antibodies).
- Optimized Mouse-on-mouse Immunohistochemical Detection of Mouse ESR2 Proteins with PPZ0506 Monoclonal Antibody. Acta histochemica et cytochemica 2022 — PMC9631985.
- Optimized selection of specific antibodies against rodent ESR1 proteins and their application for immunohistochemistry and dual immunohistofluorescence with the specific anti-ESR2 antibody PPZ0506. Histochemistry and cell biology 2026 — PMC12811342.
- Sex and interspecies differences in ESR2-expressing cell distributions in mouse and rat brains. Biology of sex differences 2023 — PMC10726529.
- Estrogen Receptor Type 1 and Type 2 Presence in Paravertebral Skeletal Muscles: Expression Level and Relation to Phenotype in Children with Idiopathic Scoliosis. Genes 2022 — PMC9141030.
- PubMed PMID:9473491 — UniProt-cited evidence.
- PubMed PMID:9636657 — UniProt-cited evidence.
- PubMed PMID:9685228 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ESR2 (Estrogen receptor beta). Expected localisation Nuclear staining in tissue sections (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: M00786-1 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Cells in zona glomerulosa); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human cervix cancer tissue; fixative not specified (datasheet M00786-1); 2. Fixation: Image fixative and duration unreported (datasheet M00786-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit monoclonal (clone IDI-5) anti-ESR2, 1:50 (datasheet M00786-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Weak staining and medium antibody–RNA consistency (HPA tissue IHC); Isoform / epitope: 9 isoforms; verify epitope coverage (UniProt); Regulation: Expression varies by tissue (UniProt). Evidence: UniProt Q92731, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to…, Datasheet M00786-1, PMC9631985, PMC10726529. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ESR2/immunohistochemistry-ihc-guide.