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RNF125 / E3 ubiquitin-protein ligase RNF125 · IHC design guide
Design Immunohistochemistry for RNF125
Plan RNF125 IHC on paraffin sections using the catalog antibody at 1:100–1:300 (datasheet A08878). Assess cytoplasmic and nuclear staining while accounting for uncertain agreement between antibody staining and RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RNF125 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 | Cytoplasmic and nuclear staining in tissue (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic and nuclear signal in several tissues (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A08878) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+3 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 | Low agreement between antibody staining and RNA expression (HPA tissue IHC) | |
| Regulation | Preferential expression in CD4+ and CD8+ T cells (UniProt) | |
| Isoform / epitope | No isoforms annotated; chain spans aa 2–232 (UniProt) |
Section 1
Recommended RNF125 IHC & IF Protocols
The catalog antibody protocol uses heat-mediated Tris-EDTA pH 9.0 retrieval (datasheet A08878). These tables summarize two published RNF125 IHC protocols (PMC12075997; PMC5564816).
| Sample | Paraffin-embedded human Gastric adenocarcinoma tissue; fixative not specified (datasheet A08878) |
| Fixation | Image fixative and duration unreported (datasheet A08878); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A08878); 20 min, 95–100 °C (standard) |
| 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 anti-RNF125, 1:100-1:300 (datasheet A08878) |
| 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 | RNF125-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic and nuclear expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 retrieval for the catalog antibody (datasheet A08878); neither article specifies a retrieval buffer (PMC12075997; PMC5564816).
Section 2
What Is the Expected RNF125 Staining Pattern?
RNF125 is mainly associated with the Golgi apparatus and can show a reticular intracellular pattern; UniProt reports no transmembrane segment and no plasma membrane detection (UniProt Q96EQ8). Expression is predominant in CD4(+) and CD8(+) T cells (UniProt Q96EQ8). HPA reports cytoplasmic and nuclear tissue staining, but rates its IHC pattern Uncertain because antibody staining has low consistency with RNA expression (HPA tissue IHC).
| Intracellular cytoplasmic staining, especially around the Golgi region, in a lymphoid cell population. | This fits the reported Golgi and reticular localization and the preference for T cells (UniProt Q96EQ8). Treat morphology as supportive, not confirmation: HPA rates its tissue IHC pattern Uncertain (HPA tissue IHC). |
| Strong staining confined to cell borders, with little intracellular signal. | A surface-only pattern conflicts with the report that RNF125 is not detected at the plasma membrane (UniProt Q96EQ8). Reassess the chromogenic signal and antibody specificity before scoring these cells as positive; compartment alone cannot establish the cause (standard IHC practice). |
| Prominent staining in a cell type outside the expected lymphoid population. | UniProt describes preferential expression in T cells, while HPA reports Medium staining in several nonlymphoid cells, including colon glandular cells (UniProt Q96EQ8; HPA tissue IHC). Unexpected staining warrants checks for cross-reactivity or endogenous detection activity; it is not automatically false (standard IHC practice). |
| Even chromogen across tissue, stroma, and areas lacking recognizable cellular localization. | A diffuse deposit is difficult to reconcile with the reported intracellular distribution (UniProt Q96EQ8). Consider nonspecific background or endogenous detection activity and compare with a negative reagent control before assigning RNF125 positivity (standard IHC practice). |
| No staining in a section selected because HPA reports Medium signal there. | For example, HPA lists adrenal gland glandular cells and appendix glandular cells at Medium intensity (HPA tissue IHC). Absence prompts review of tissue identity, controls, and assay performance; HPA’s Uncertain IHC reliability means either tissue is a provisional reference, not a guaranteed positive control (HPA tissue IHC; standard IHC practice). |
💡Expected RNF125 appearanceA plausible positive IHC result is intracellular cytoplasmic, often Golgi-associated staining in T-cell-rich areas (UniProt Q96EQ8), with intensity assessed against local controls because HPA’s tissue IHC is Uncertain (HPA tissue IHC); uniform background or surface-only staining should trigger review (UniProt Q96EQ8; standard IHC practice).
| Cell population used for interpretation | UniProt reports predominant expression in lymphoid tissues, including bone marrow, spleen, and thymus, and a preference for CD4(+) and CD8(+) T cells (UniProt Q96EQ8). HPA reports tissue-enhanced RNA expression in bone marrow but does not provide a dependable IHC-positive T-cell control (HPA tissue IHC). |
| Reported compartment versus tissue staining | Golgi localization is supported in HPA ICC-IF, whereas additional nucleolar localization is Uncertain (HPA subcellular). HPA tissue IHC describes cytoplasmic and nuclear expression but assigns the overall pattern Uncertain reliability (HPA tissue IHC). Interpret nuclear IHC cautiously rather than treating it as independently confirmed localization. |
| Antibody evidence | For HPA041514, IHC is rated Uncertain and ICC is rated Approved (HPA antibodies). Approval in ICC does not resolve the stated low consistency between tissue antibody staining and RNA expression in IHC (HPA antibodies; HPA tissue IHC). |
| IF/ICC Q: What localization should be expected? | A: Mainly Golgi-associated signal is supported; nucleolar signal is an additional Uncertain localization (HPA subcellular). HPA lists ICC-IF images from A-431, SK-MEL-30, and U2OS, but those images do not establish a paraffin IHC protocol or validate tissue IHC staining (HPA subcellular; HPA antibodies). |
| Situation | Likely cause | Next action |
|---|---|---|
| The entire section has weak or absent signal. | The staining run may have failed, or the chosen tissue may have little detectable signal; UniProt reports weaker expression outside predominantly lymphoid tissues (UniProt Q96EQ8; standard IHC practice). | Check the run’s reagent and detection controls, then compare a documented HPA Medium staining site such as adrenal gland glandular cells; interpret a negative result cautiously because HPA tissue IHC reliability is Uncertain (HPA tissue IHC; standard IHC practice). |
| Signal appears mainly at the plasma membrane. | This distribution conflicts with UniProt’s report of intracellular membranes and no detected plasma membrane localization; the image alone does not identify the artefact (UniProt Q96EQ8). | Inspect adjacent morphology and negative reagent controls for edge deposits or nonspecific staining, and require convincing intracellular signal before calling the result consistent with RNF125 localization (UniProt Q96EQ8; standard IHC practice). |
| Nuclear staining dominates while cytoplasmic staining is scarce. | HPA reports nuclear as well as cytoplasmic tissue IHC, but its tissue pattern is Uncertain; HPA’s additional nucleolar ICC-IF location is also Uncertain (HPA tissue IHC; HPA subcellular). | Record nuclear and cytoplasmic scores separately and compare with controls; avoid presenting dominant nuclear IHC as confirmed RNF125 localization on these sources alone (HPA tissue IHC; HPA subcellular; standard IHC practice). |
| Brown signal covers stroma and multiple cell types without clear intracellular detail. | Diffuse staining can arise from background or endogenous detection activity in chromogenic IHC; it does not show the reported Golgi-associated intracellular distribution (standard IHC practice; UniProt Q96EQ8). | Review the negative reagent control, blocking and wash performance, and tissue morphology before scoring; interpret only localized cellular staining above control background (standard IHC practice). |
| Unexpected nonlymphoid cells stain strongly. | T-cell preference does not imply exclusive expression: HPA reports Medium staining in several nonlymphoid cell types, although its IHC reliability is Uncertain (UniProt Q96EQ8; HPA tissue IHC). | Document the exact cell type and compartment, compare the negative reagent control, and seek independent antibody or orthogonal evidence before attributing a surprising pattern to RNF125 (HPA tissue IHC; standard IHC practice). |
| A tissue expected to be negative shows staining. | HPA lists cardiomyocytes in heart muscle and cholangiocytes in liver as Not detected, but its tissue IHC pattern has Uncertain reliability (HPA tissue IHC). | Confirm the stained cell type and compare controls; report the observation without using a single HPA negative entry to declare cross-reactivity or RNF125 expression (HPA tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing RNF125 staining
A08878 · IHCImmunohistochemical analysis of paraffin-embedded human Gastric adenocarcinoma. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDT
A30664 · IHCImmunohistochemical analysis of paraffin-embedded human Gastric adenocarcinoma. 1, Antibody was diluted at 1:200(4° overnight). 2, Tris-EDTA
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000101695-RNF125/tissue/cerebral+cortex#imid_10650217
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000101695-RNF125/tissue/colon#imid_10650143
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000101695-RNF125/tissue/liver#imid_10650136
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000101695-RNF125/tissue/pancreas#imid_10650079
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA041514; green = RNF125, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA041514; green = RNF125, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RNF125 IHC & IF
🧪Run adrenal gland first: glandular cells should stain for RNF125 (HPA: Medium in adrenal gland glandular cells). Use parathyroid gland as the negative tissue (HPA: Not detected in parathyroid gland glandular cells); on the adrenal slide, assess non-glandular stromal areas as background references, without assuming every stromal cell is RNF125-negative.
Positive control tissue: Adrenal gland (Glandular cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RNF125 in A-431, SK-MEL-30, U2OS, with annotated localisation: Golgi apparatus (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species, immunoglobulin class, and clonality, and RNF125 knockout material if available. For chromogenic adrenal IHC, quench endogenous peroxidase and check vascular areas for residual background (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A08878 tissue-IHC caption does not state the fixative (A08878 caption: fixative not stated). The documented paraffin-section example uses Tris-EDTA retrieval at pH 9.0 and a 1:200 primary dilution; it does not establish that other retrieval conditions will work (A08878 caption). Paraffin IHC has a documented starting protocol, while no frozen-section comparison is supplied; ICC-IF images offer a way to check Golgi localisation in A-431, SK-MEL-30, or U2OS cells (HPA subcellular: Golgi supported; HPA: ICC-IF image cell lines).
HPA tissue IHC evidence for RNF125
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low 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 | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Adipocytes | Medium | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RNF125 IHC Tips
Troubleshoot RNF125 staining in paraffin sections by checking retrieval, cellular context and compartment before comparing chromogenic signal across samples.
Which retrieval conditions should I use when RNF125 staining is weak?
Use heat-mediated Tris-EDTA at pH 9.0 for RNF125 paraffin sections (datasheet A08878). The selected image used this retrieval with primary antibody at 1:200 overnight at 4°C, but its caption does not state the fixative (datasheet A08878). If staining remains weak, compare a small retrieval-time series while keeping section thickness, antibody incubation and chromogen development constant (standard IHC practice). A citrate buffer at pH 6.0 is a fallback to test alongside the documented condition, not an established RNF125 requirement (standard IHC practice; datasheet A08878).
How should I troubleshoot RNF125 staining after tissue fixation?
RNF125-specific sensitivity to fixation is unknown: the selected paraffin-section caption does not report a fixative, and the supplied evidence does not establish a target-specific fixation effect (datasheet A08878). Record the actual fixative and fixation duration for each specimen, then compare sections processed together under the documented Tris-EDTA pH 9.0 retrieval condition (standard IHC practice; datasheet A08878). If staining varies between blocks, inspect tissue preservation and compare internal cell populations before changing antibody concentration (standard IHC practice). Do not attribute a weak result to RNF125 topology or modification status without a controlled fixation comparison (UniProt Q96EQ8 topology and annotations; standard IHC practice).
Where should convincing RNF125 chromogenic staining appear within cells?
Assess RNF125 primarily as intracellular signal: UniProt describes Golgi membrane association and a reticular pattern, with no plasma-membrane detection (UniProt Q96EQ8 subcellular location). Supported Golgi localisation in cell imaging strengthens a perinuclear interpretation, whereas reported nucleolar localisation is uncertain (HPA subcellular). Chromogenic sections cannot resolve a Golgi network as clearly as cell imaging, so score cytoplasmic or perinuclear staining against cell morphology and nearby background (standard IHC practice). Treat a sharp plasma-membrane rim or isolated nuclear deposit cautiously, and check adjacent sections and negative controls before calling it RNF125 (UniProt Q96EQ8 subcellular location; standard IHC practice).
Could an isoform or hidden epitope explain inconsistent RNF125 staining?
The supplied RNF125 record annotates 0 isoforms and no glycosylation sites or modified residues, but does not identify this antibody’s epitope (UniProt Q96EQ8 annotations; datasheet A08878). Consequently, an isoform-specific or modification-specific staining explanation is unsupported by these payloads (UniProt Q96EQ8 annotations; datasheet A08878). Compare serial sections using the documented Tris-EDTA pH 9.0 retrieval and a controlled retrieval-time series to test epitope accessibility (datasheet A08878; standard IHC practice). If the pattern remains inconsistent, use an independently validated antibody or orthogonal expression evidence before assigning a molecular cause (standard IHC practice).
How can I check RNF125 localisation with multiplex immunofluorescence?
Use IF as a separate validation experiment alongside chromogenic IHC, pairing RNF125 with a T-cell marker such as CD3 when examining lymphoid tissue (UniProt Q96EQ8 tissue specificity; standard IF practice). Select spectrally separated fluorophores and place the weaker signal in a channel with low measured tissue autofluorescence (standard IF practice). RNF125 has no annotated transmembrane segment, but its epitope and membrane-facing accessibility are unspecified; titrate permeabilisation against the epitope’s demonstrated accessibility rather than assuming a membrane side (UniProt Q96EQ8 topology; datasheet A08878; standard IF practice). Compare perinuclear signal with a Golgi marker, and regard nucleolar overlap as provisional because that additional location is uncertain (HPA subcellular; standard IF practice).
What causes diffuse or punctate background in RNF125 IHC?
Run a no-primary control and inspect unstained tissue pigment before interpreting diffuse or punctate chromogen as RNF125 (standard IHC practice). For peroxidase-based detection, include a peroxidase block and check whether residual endogenous enzyme or excess DAB development tracks the background (standard IHC practice). The selected paraffin-section image used primary antibody at 1:200 overnight at 4°C and secondary antibody at 1:200 for 45 minutes at room temperature (datasheet A08878). Titrate detection and washing around those documented conditions, then favor reproducible intracellular staining over deposits in tissue folds, damaged edges or necrotic areas (datasheet A08878; standard IHC practice).
How should I score RNF125 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because RNF125 is predominantly reported in lymphoid tissues and T cells, while tissue IHC also reports other stained cell types (UniProt Q96EQ8 tissue specificity; HPA tissue IHC). For intensity comparisons, report an H-score from 0–300 or the percentage of positive cells with a prespecified threshold (standard IHC practice). For infiltrates, report positive-cell density per mm² and normalise to viable tissue area or the relevant cell population (standard IHC practice). Keep retrieval, imaging, threshold and exclusion of folds or necrosis consistent, and disclose the uncertain HPA tissue-IHC reliability (datasheet A08878; HPA tissue IHC; standard IHC practice).
How can I distinguish true RNF125 staining from artefact?
A convincing result should recur in comparable intracellular compartments and plausible cells across intact sections, with low signal in matched negative controls (UniProt Q96EQ8 subcellular location and tissue specificity; standard IHC practice). A crisp plasma-membrane outline conflicts with the reported absence of plasma-membrane detection, while nucleolar staining has only uncertain support (UniProt Q96EQ8 subcellular location; HPA subcellular). Check edge-concentrated signal, necrotic debris and staining retained in a no-primary control for section or endogenous-enzyme artefacts (standard IHC practice). Interpret apparent tissue specificity cautiously because HPA rates its RNF125 tissue staining Uncertain owing to low consistency with RNA expression (HPA tissue IHC).
Boster reagents
Best RNF125 / E3 ubiquitin-protein ligase RNF125 IHC Antibodies
Both anti-RNF125 antibodies have paraffin-section IHC images and list IF and human, mouse, and rat reactivity (catalog: A08878 and A30664 applications, reactivity, IHC image captions).
Real IHC data
Anti-TRAC-1 RNF125 Antibody
Cat # A08878
Real IHC data
Anti-RNF125 Antibody
Cat # A30664
A08878 lists IHC and IF for human, mouse, and rat; its IHC image shows paraffin-embedded human gastric adenocarcinoma (catalog: A08878 applications, reactivity, IHC image caption). A30664 lists IHC, IF, and ICC for the same species; its IHC image also shows paraffin-embedded human gastric adenocarcinoma (catalog: A30664 applications, reactivity, IHC image caption).
Which to pick: For paraffin-section tissue IHC, either SKU has its own image using 1:200 primary antibody and Tris-EDTA retrieval at pH 9.0; neither caption reports the fixative (catalog: A08878 and A30664 IHC image captions). For IF/ICC, choose A30664 if ICC is required because it lists both applications and is described as polyclonal; A08878 lists IF but does not list ICC (catalog: A30664 applications and dilution_raw; A08878 applications). For cross-species work, both list human, mouse, and rat reactivity, while their supplied IHC images show human tissue (catalog: A08878 and A30664 reactivity, IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q96EQ8 (RN125_HUMAN, E3 ubiquitin-protein ligase RNF125).
- Human Protein Atlas. RNF125 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. RNF125 subcellular location (ICC-IF): Mainly localized to the Golgi apparatus. In addition localized to the nucleoli..
- Human Protein Atlas. RNF125 antibody validation summary (1 antibodies).
- Hypermethylation of RNF125 promotes autophagy-induced oxidative stress in asthma by increasing HMGB1 stability. iScience 2023 — PMC10432822.
- RNA‑binding protein MBNL1 regulates tumor growth, chemosensitivity and antitumor immunity in lung adenocarcinoma by controlling the expression of tumor suppressor RNF125. Oncology reports 2025 — PMC12075997.
- Ubiquitin ligase RNF125 targets PD-L1 for ubiquitination and degradation. Frontiers in oncology 2022 — PMC9374197.
- Ring finger protein 125, as a potential highly aggressive and unfavorable prognostic biomarker, promotes the invasion and metastasis of human gallbladder cancers via activating the TGF- β1-SMAD3-ID1 signaling pathway. Oncotarget 2017 — PMC5564816.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:12974981 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RNF125 (E3 ubiquitin-protein ligase RNF125). Expected localisation Cytoplasmic and nuclear staining in tissue (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A08878). Validated IHC antibody: A08878 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human Gastric adenocarcinoma tissue; fixative not specified (datasheet A08878); 2. Fixation: Image fixative and duration unreported (datasheet A08878); 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 retrieval: Tris-EDTA pH 9.0 (datasheet A08878); 20 min, 95–100 °C (standard); 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 anti-RNF125, 1:100-1:300 (datasheet A08878); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Low agreement between antibody staining and RNA expression (HPA tissue IHC); Isoform / epitope: No isoforms annotated; chain spans aa 2–232 (UniProt); Regulation: Preferential expression in CD4+ and CD8+ T cells (UniProt). Evidence: UniProt Q96EQ8, HPA tissue IHC · Uncertain, HPA ICC-IF · Mainly localized to the Golgi apparatus. In addition localized to…, Datasheet A08878, PMC12075997, PMC5564816. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RNF125/immunohistochemistry-ihc-guide.