This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
ESR2 / Estrogen receptor beta · IHC design guide
Design Immunohistochemistry for ESR2
Plan ESR2 chromogenic IHC around the low nuclear staining reported in testis and ovary (HPA tissue IHC). Use consistent fixation and nuclear scoring to interpret weak signals.
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 | Low nuclear tissue staining (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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Low signal; staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | RNA enhanced: adrenal, ovary, testis (HPA tissue RNA) | |
| Isoform / epitope | 9 isoforms; confirm antibody epitope coverage (UniProt) |
Section 1
Recommended ESR2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by one published ESR2 protocol for mouse and rat brain sections (PMC10726529).
| 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 rule: nuclear antigen). The published brain protocol uses citrate pH 6.0 (PMC10726529).
Section 2
What Is the Expected ESR2 Staining Pattern?
ESR2 is a nuclear receptor without a transmembrane segment (UniProt Q92731: subcellular location and topology). In paraffin section IHC, expect chiefly nuclear staining; HPA reports low nuclear expression across several tissues, including testis and ovary, and medium staining in adrenal zona glomerulosa cells (HPA: tissue IHC). HPA rates its tissue profile Enhanced while describing medium consistency between antibody staining and RNA expression (HPA: tissue IHC reliability).
| Nuclear staining in adrenal zona glomerulosa cells. | This matches the reported medium IHC signal in those cells and ESR2's nuclear location (HPA: tissue IHC; UniProt Q92731: subcellular location). Judge intensity against the same run's background and controls (standard IHC practice). |
| Faint nuclear staining in Leydig cells or ovarian stroma cells. | Low signal can fit the reported pattern in these specific cells (HPA: tissue IHC). A weak result here alone should not be scored as assay failure; assess nuclear definition and the positive control (standard IHC practice). |
| Predominantly cytoplasmic or membrane staining with little nuclear signal. | This conflicts with the expected IHC pattern and UniProt's nuclear assignment (HPA: tissue IHC; UniProt Q92731: subcellular location). Check staining specificity and processing before interpreting it as ESR2; compartment alone cannot identify the cause (standard IHC practice). |
| Signal in a cell population listed as not detected, such as adipocytes in adipose tissue. | HPA reports no detected staining in that sampled cell type, so investigate cross-reactivity or endogenous chromogenic activity (HPA: tissue IHC; standard IHC practice). The finding does not establish that every cell in the tissue lacks ESR2. |
| Diffuse slide background, or no signal in adrenal zona glomerulosa cells. | Diffuse colour obscures nuclear scoring; inspect blocking, washes and detection controls (standard IHC practice). Absent signal in the reported medium-staining population calls for a positive-control and assay check, but one section cannot establish target absence (HPA: tissue IHC; standard IHC practice). |
💡Expected ESR2 appearanceCall a section positive when staining is predominantly nuclear in an appropriate cell population, with medium signal possible in adrenal zona glomerulosa cells and low signal reported in Leydig or ovarian stroma cells; diffuse background or isolated non-nuclear colour is suspect (HPA: tissue IHC; UniProt Q92731: subcellular location; standard IHC practice).
| Cell population and reference intensity | HPA reports medium staining in adrenal zona glomerulosa cells but low staining in Leydig cells and ovarian stroma cells (HPA: tissue IHC). Compare like cell populations when setting an expected intensity; a whole-organ label does not identify which cells should stain. |
| Subcellular location | UniProt assigns ESR2 to the nucleus and reports no transmembrane segment (UniProt Q92731: subcellular location and topology). Nuclear localisation is therefore the primary IHC interpretation criterion; membrane colour needs independent validation before being called target signal. |
| Isoforms and antibody recognition | UniProt lists nine ESR2 isoforms (UniProt Q92731: isoforms 1–9). That establishes sequence variation, but the supplied record gives no epitope or isoform coverage for a chosen antibody; check its documented recognition before attributing a staining difference to an isoform. |
| Strength of antibody evidence | The tissue profile is Enhanced, with medium agreement between staining and RNA; the listed mouse monoclonal has Supported IHC status (HPA: tissue IHC reliability; HPA: CAB079300 validation). These labels support use as references, while a new assay still needs its own controls (standard IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| No nuclear signal in the adrenal zona glomerulosa positive reference. | This conflicts with HPA's medium staining report, though a single negative section does not prove target absence (HPA: tissue IHC). | Confirm the relevant cells are present, then review the IHC-validated antibody's documented retrieval and detection conditions and the run's positive control (standard IHC practice). |
| Testis or ovary appears nearly negative. | The reported signal is low in Leydig cells and ovarian stroma cells, so a whole-section impression may miss sparse weak nuclei (HPA: tissue IHC). | Inspect the specified cell population at suitable magnification and compare it with a same-run reference; avoid calling the organ uniformly negative (HPA: tissue IHC; standard IHC practice). |
| Colour lies mainly outside nuclei. | Predominantly non-nuclear IHC conflicts with ESR2's nuclear assignment; staining location alone cannot distinguish nonspecific binding from detection artefact (UniProt Q92731: subcellular location; standard IHC practice). | Examine a reagent-control section and the positive reference, then reassess blocking and detection before scoring the colour as ESR2 (standard IHC practice). |
| Unexpected signal appears in adipocytes from adipose tissue. | HPA lists those cells as not detected; cross-reactivity or endogenous chromogenic activity is possible (HPA: tissue IHC; standard IHC practice). | Compare the same cell type with an appropriate detection control and review blocking; report the discrepancy at cell level (standard IHC practice). |
| Diffuse colour prevents a clear nuclear call. | Background can arise from nonspecific reagent binding, inadequate washes or endogenous detection activity (standard IHC practice). | Use reagent controls to locate the background source, then review blocking, wash steps and chromogenic development before rescoring (standard IHC practice). |
| What should an IF/ICC image show? | HPA supports nucleoplasmic localisation and marks additional vesicle localisation uncertain; its IF evidence does not redefine the primary IHC pattern (HPA: subcellular ICC-IF). | Expect nucleoplasmic signal as the main localisation; treat vesicle signal cautiously and evaluate it within the separate IF/ICC guide (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 medium ESR2 staining (HPA: adrenal gland, cells in zona glomerulosa, Medium). Use adipose tissue adipocytes as the negative comparison (HPA: adipose tissue, adipocytes, Not detected); cells on the adrenal slide without nuclear signal can serve as internal background references, but their ESR2-negative status is unverified (UniProt Q92731: nucleus; HPA: adrenal gland, cells in zona glomerulosa, Medium).
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, an isotype control matched to the primary antibody’s host species and clonality, and a biological negative such as an ESR2 knockout or validated peptide block (standard IHC practice). For adrenal chromogenic IHC, quench endogenous peroxidase and block endogenous biotin if using biotin-based detection; check background on the same tissue (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and retrieval effect are unreported in the supplied evidence; optimize antigen retrieval empirically for paraffin sections (selected-SKU tissue-IHC caption: paraffin-embedded human cervix cancer; standard IHC practice). The exact M00786-1 tissue-IHC caption does not state a fixative, so its fixative is unreported (selected-SKU tissue-IHC caption). ICC-IF images exist for RT-4, SK-MEL-30 and U2OS, but the supplied evidence does not establish that tissue IF or frozen sections are easier, or document a specific adrenal artefact (HPA subcellular: cell lines with ICC-IF images; supplied target/application evidence).
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 in paraffin sections by checking nuclear signal, cell identity, antibody specificity and matched controls.
What retrieval should I use when ESR2 nuclear staining is weak?
Use Tris-EDTA at pH 9.0 for heat-induced retrieval at 95–98 °C for 20 min (page protocol: ESR2 IHC retrieval). Let slides cool in retrieval buffer, then compare staining with a slide processed identically except for retrieval; consistent handling makes the comparison interpretable (standard IHC practice). Score nuclei in the expected cell population because ESR2 is nuclear and HPA reports low nuclear expression in several tissues (UniProt Q92731: nucleus; HPA: tissue IHC profile). If staining remains weak, test citrate at pH 6.0 as a fallback on matched sections, keeping detection and exposure to chromogen constant (standard IHC practice).
How can I troubleshoot ESR2 staining across differently fixed paraffin samples?
Target-specific sensitivity of ESR2 staining to fixation is unknown from the supplied evidence; record the fixative, fixation duration and processing history for each specimen before comparing results (supplied IHC evidence; standard IHC practice). The catalog image shows staining in paraffin-embedded human cervix cancer, but its caption does not state a fixative (catalog caption: M00786-1). Run matched sections through the same Tris-EDTA pH 9.0, 95–98 °C, 20 min retrieval and detection steps (page protocol: ESR2 IHC retrieval; standard IHC practice). Compare nuclear staining with a control section processed alongside each batch, and report any fixation differences without assigning them an ESR2-specific effect (UniProt Q92731: nucleus; standard IHC practice).
Should cytoplasmic or vesicular staining count as ESR2 positive in chromogenic IHC?
Prioritize nuclear staining when calling ESR2 positive: UniProt places ESR2 in the nucleus, and HPA describes low nuclear expression across several tissues (UniProt Q92731: subcellular location; HPA: tissue IHC profile). HPA supports a nucleoplasmic location in cell imaging but labels its additional vesicular location uncertain (HPA: subcellular summary). Record cytoplasmic or punctate chromogen separately from the nuclear score, especially when nuclei in the same section are unstained (standard IHC practice). Check morphology, counterstain and the no-primary control before interpreting such signal; a possible vesicular location alone does not establish that chromogenic deposits represent ESR2 (HPA: subcellular summary; standard IHC practice).
How do ESR2 isoforms and epitope placement affect an IHC result?
ESR2 has 9 listed isoforms, so determine which sequence region the IHC-validated antibody recognizes before treating a negative section as absence of all ESR2 (UniProt Q92731: isoforms; standard IHC practice). Map the stated epitope against the isoforms and the ligand-binding domain at residues 264–498 (UniProt Q92731: isoforms and NR LBD). UniProt also lists phosphoserines at residues 87 and 105; their presence does not establish that this antibody is modification sensitive (UniProt Q92731: modified residues; standard IHC practice). Compare matched positive and no-primary control sections, and report the antibody's documented epitope coverage alongside any nuclear staining score (standard IHC practice; UniProt Q92731: nucleus).
How should I cross-check an ESR2 IHC pattern by multiplex IF?
Use IF as a separate validation experiment and include a marker identifying the cell population being scored; HPA reports medium staining in adrenal zona glomerulosa cells and low staining in ovarian stroma and testis Leydig cells (HPA: tissue IHC). Select fluorophores away from the tissue's strongest autofluorescence and inspect single-label controls before interpreting overlap (standard IF practice). ESR2 has no transmembrane segment and is principally nucleoplasmic, so use a mild permeabilisation condition that permits nuclear access, then verify that nuclei retain clear morphology (UniProt Q92731: topology and nucleus; HPA: supported nucleoplasm; standard IF practice). Keep IF localisation and chromogenic IHC scores separate because fluorescence and chromogen have different background sources (standard IHC/IF practice).
What should I check when ESR2 chromogen appears throughout the section?
First inspect a no-primary control and confirm that endogenous peroxidase blocking was effective before assigning diffuse DAB deposits to ESR2 (standard chromogenic IHC practice). Check whether stronger deposits track tissue edges, damaged areas or nonspecific surface staining rather than intact nuclei, the expected principal compartment (standard IHC practice; UniProt Q92731: nucleus). Compare an antibody dilution series on matched sections while keeping retrieval at pH 9.0, 95–98 °C for 20 min and DAB development consistent (page protocol: ESR2 IHC retrieval; standard IHC practice). If background persists, review blocking, washes and detection controls before interpreting weak nuclear signal in tissues where HPA reports low expression (standard IHC practice; HPA: tissue IHC profile).
How should I score patchy ESR2 nuclear staining across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and count only interpretable nuclei within comparable tissue regions, since ESR2 is predominantly nuclear and HPA reports cell-specific staining levels (UniProt Q92731: nucleus; HPA: tissue IHC). Record the percentage of nuclei at each intensity from 0–3, then calculate an H-score from 0–300; also retain the percentage of positive nuclei (standard IHC scoring practice). Normalize each score to the number of eligible nuclei examined, and report sampled area or cell count so unequal specimen size is visible (standard IHC scoring practice). Use identical retrieval, chromogen development and scoring thresholds across batches, with a processed control section to detect staining drift (standard IHC practice).
How can I distinguish convincing ESR2 staining from artefact?
A convincing result shows reproducible nuclear staining in morphologically intact cells with appropriate control behavior, consistent with ESR2's nuclear location (UniProt Q92731: nucleus; standard IHC practice). For context, HPA reports medium staining in adrenal zona glomerulosa cells, low staining in several other listed cell populations and no detection in several listed populations; evaluate the particular cell type, not just the tissue name (HPA: tissue IHC). Treat edge-only deposits, necrotic regions, predominantly cytoplasmic staining and signal in a no-primary control as reasons to investigate artefact (standard IHC practice; UniProt Q92731: nucleus). Check endogenous peroxidase controls before assigning DAB signal to ESR2, and describe discordant patterns without assuming that every stained cell expresses the receptor (standard chromogenic IHC practice).
Boster reagents
Best ESR2 / Estrogen receptor beta IHC Antibodies
M00786-1 has an IHC image from paraffin-embedded human cervix cancer; human is the listed reactive species, and no IF image is supplied (catalog IHC image caption; reactivity; IF image alts).
Real IHC data
Anti-Estrogen Receptor beta ESR2 Rabbit Monoclonal Antibody
Cat # M00786-1
M00786-1 is listed for human IHC and WB (catalog applications; reactivity). Its own IHC caption describes paraffin-embedded human cervix cancer (catalog IHC image caption).
Which to pick: For tissue IHC, choose M00786-1: it is a rabbit monoclonal, clone IDI-5, listed for human IHC with a paraffin-section image (catalog host; clone; applications; reactivity; IHC image caption). The image caption does not report the fixative (catalog IHC image caption). No SKU in the payload is listed for IF/ICC or nonhuman reactivity, so there is no supported IF/ICC or cross-species pick (catalog applications; reactivity).
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 Low nuclear tissue staining (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 paraffin sections. (standard IHC practice; not target-specific); Caveat: Low signal; staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 9 isoforms; confirm antibody epitope coverage (UniProt); Regulation: RNA enhanced: adrenal, ovary, testis (HPA tissue RNA). Evidence: UniProt Q92731, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to…, Datasheet M00786-1, PMC10726529. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ESR2/immunohistochemistry-ihc-guide.