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RBMS3 / RNA-binding motif, single-stranded-interacting protein 3 · IHC design guide
Design Immunohistochemistry for RBMS3
This guide helps plan chromogenic RBMS3 IHC in paraffin sections using the reported cytoplasmic tissue pattern (HPA tissue IHC). Use high staining in breast glandular cells as a reference, and interpret scores cautiously because tissue staining reliability is uncertain (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RBMS3 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 staining in several tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic signal in breast glandular cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A12416-3) | |
| Positive control | Breast+4 more · see all | |
| Negative control | Adrenal gland+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across compared paraffin sections. (selected-SKU IHC image A12416-3) | |
| Caveat | Tissue staining has low concordance with RNA data (HPA tissue IHC) | |
| Regulation | Low tissue specificity at RNA level (HPA tissue RNA) | |
| Isoform / epitope | 5 isoforms; epitope coverage needs checking (UniProt) |
Section 1
Recommended RBMS3 IHC & IF Protocols
The catalog antibody uses citrate pH 6 heat retrieval (datasheet: A12416-3). The published RBMS3 IHC protocols below cover breast, gallbladder, gastric and ovarian tissues (PMC9917836; PMC7251710; PMC5352053; PMC11079397).
| Sample | Paraffin-embedded human oesophagus squama cancer tissues; fixative not specified (datasheet A12416-3) |
| Fixation | Image fixative and duration unreported (datasheet A12416-3); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A12416-3) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A12416-3) |
| Primary antibody | Rabbit anti-RBMS3, 0.5-1μg/ml (datasheet A12416-3) |
| Primary incubation | Overnight at 4 °C (datasheet A12416-3) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A12416-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RBMS3-positive staining in glandular cells of breast (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval (datasheet: A12416-3); the gallbladder and gastric protocols specify temperatures and times for this buffer (PMC7251710; PMC5352053).
Section 2
What Is the Expected RBMS3 Staining Pattern?
RBMS3 is annotated as cytoplasmic and has no transmembrane segment (UniProt Q6XE24). In paraffin tissue IHC, expect cytoplasmic staining in selected cell populations, including cerebellar Purkinje cells, cerebral cortex neurons and breast glandular cells (HPA: High in each). Treat the pattern as provisional: HPA rates tissue IHC reliability Uncertain because antibody staining has low consistency with RNA expression (HPA: Uncertain).
| Cytoplasmic staining in cerebellar Purkinje cells or cerebral cortex neurons, with little nuclear signal. | This matches the annotated compartment (UniProt Q6XE24: cytoplasm) and reported High staining in those cell populations (HPA: tissue IHC). Compare cell types on the same section; HPA's Uncertain rating means a plausible pattern alone does not establish antibody specificity. |
| Predominantly nuclear staining, with little or no cytoplasmic staining in the expected cells. | A predominantly nuclear IHC pattern conflicts with the cytoplasmic annotation and HPA tissue profile (UniProt Q6XE24: cytoplasm; HPA: cytoplasmic expression). Review morphology, counterstain and controls before treating it as RBMS3 localisation; the supplied sources do not establish a nuclear IHC pattern. |
| Strong staining in cells reported as Not detected, such as esophageal squamous epithelial cells. | This departs from the reported tissue IHC pattern (HPA: Not detected in esophageal squamous epithelial cells). Cross-reactivity or endogenous detection activity is possible; compare a no-primary control and a reported positive tissue before assigning the signal to RBMS3. |
| Diffuse color across tissue, including extracellular spaces or cells expected to be unstained. | Widespread haze obscures the cell-specific cytoplasmic pattern (HPA: cytoplasmic expression in several tissues). In general IHC practice, excess antibody, incomplete blocking or residual detection activity can contribute; assess background controls before scoring cellular signal. |
| No staining in a reported High population, such as kidney glomerular cells. | A blank result fails to reproduce a reported High IHC observation (HPA: kidney cells in glomeruli). Check tissue integrity, staining controls and the validated IHC workflow; because HPA rates this tissue profile Uncertain, one negative section cannot by itself resolve expression or assay failure. |
💡Expected RBMS3 appearanceCall a section provisionally positive when cytoplasmic chromogen is evident in reported High populations such as cerebellar Purkinje cells (UniProt Q6XE24: cytoplasm; HPA: High in Purkinje cells), while predominant nuclear color or broad staining of HPA Not detected cell types raises concern for a false positive (HPA: tissue IHC, Uncertain).
| Compartment and topology | RBMS3 is annotated in the cytoplasm, with no transmembrane segment or signal peptide (UniProt Q6XE24). Score cellular cytoplasmic staining; these annotations do not predict an extracellular or membrane-restricted IHC pattern. |
| Cell population and tissue choice | HPA reports High staining in breast glandular cells, neurons, Purkinje cells, kidney glomerular cells and skeletal myocytes; several glandular and squamous populations are Not detected (HPA: tissue IHC). Select controls by cell type rather than whole-organ label. |
| IHC evidence strength | The listed antibody HPA034719 has Uncertain IHC status, and HPA reports low consistency between staining and RNA expression (HPA: antibody validation; tissue reliability). Treat intensity and negative calls as assay observations requiring controls, not definitive expression measurements. |
| Isoforms and antibody recognition | Five isoforms and two RRM regions are annotated (UniProt Q6XE24: isoforms 1–5; RRM 1, residues 61–134; RRM 2, residues 140–225). No antibody epitope is supplied, so isoform recognition or retrieval-dependent epitope exposure cannot be predicted. |
| Processing and fixation evidence | No signal peptide, propeptide, glycosylation sites or modified residues are annotated (UniProt Q6XE24). These facts do not establish fixation sensitivity; target-specific effects of formalin fixation or antigen retrieval are unreported in the supplied sources. |
| IF/ICC Q&A: should IHC look punctate? | HPA reports approved vesicle localisation in ICC-IF (HPA: subcellular; HPA034719 ICC Approved). For paraffin IHC, use the separate cytoplasmic tissue profile (HPA: tissue IHC, Uncertain); the IF observation does not establish a required punctate chromogenic pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| No chromogen in a reported High cell population (HPA: tissue IHC). | The staining run may have failed, or the observed section may differ from the uncertain HPA profile (HPA: tissue IHC, Uncertain). | Check a run control and tissue morphology, then review the IHC-validated antibody's documented dilution and retrieval settings where available; repeat with a reported High cell population and interpret the result cautiously (general IHC practice; HPA: tissue IHC). |
| Predominant nuclear signal in a candidate positive section. | That distribution conflicts with the cytoplasmic annotation and tissue profile (UniProt Q6XE24; HPA: tissue IHC). | Verify nuclear boundaries with the counterstain, compare a no-primary control and reassess the cellular signal before calling it RBMS3 (general IHC practice). |
| Strong color in HPA Not detected esophageal squamous cells. | The result conflicts with the reported cell-type pattern; cross-reactivity or endogenous detection activity may explain it (HPA: tissue IHC; general IHC practice). | Run a no-primary control, inspect detection-system background and compare with a reported High tissue; avoid treating the HPA negative call as absolute because tissue IHC reliability is Uncertain (HPA: tissue IHC). |
| Diffuse staining prevents identification of cytoplasmic signal. | Nonspecific antibody binding, incomplete blocking or residual detection activity can create background (general IHC practice). | Compare no-primary and positive controls, review blocking and washes, and titrate the primary antibody within its documented IHC range if available (general IHC practice). |
| Only a few cells stain in a mixed tissue section. | HPA assigns levels to named cell populations, while RBMS3 RNA has low tissue specificity (HPA: tissue IHC; HPA: RNA specificity). | Identify and score the named cells separately from adjacent populations; record localisation and intensity rather than labeling the whole section positive or negative (general IHC practice). |
| Punctate ICC-IF staining appears inconsistent with diffuse cytoplasmic IHC. | HPA reports vesicles for ICC-IF but describes cytoplasmic expression for tissue IHC; the applications have different evidence and validation status (HPA: subcellular; HPA: tissue IHC; HPA034719). | Assess each application against its own HPA pattern and controls; do not require vesicle-like puncta to accept cytoplasmic chromogenic IHC staining (HPA: ICC Approved; HPA: IHC Uncertain). |
Experiment evidence
IHC & IF images showing RBMS3 staining
A12416-3 · IHCIHC analysis of RBMS3 using anti-RBMS3 antibody (A12416-3). RBMS3 was detected in paraffin-embedded section of human oesophagus squama cance
A12416-3 · IHCIHC analysis of RBMS3 using anti-RBMS3 antibody (A12416-3). RBMS3 was detected in paraffin-embedded section of mouse brain tissues. Heat med
A12416-3 · IFIF analysis of RBMS3 using anti-RBMS3 antibody (A12416-3). RBMS3 was detected in immunocytochemical section of U20S cell. Enzyme antigen ret
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000144642-RBMS3/tissue/cerebral+cortex#imid_8785108
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000144642-RBMS3/tissue/colon#imid_8785034
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000144642-RBMS3/tissue/liver#imid_8785027
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000144642-RBMS3/tissue/kidney#imid_8785036
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA034719; green = RBMS3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA034719; green = RBMS3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RBMS3 IHC & IF
🧪Run kidney first: cells in glomeruli should stain strongly (HPA: High in cells in glomeruli). Use adrenal gland glandular cells as the negative tissue (HPA: Not detected in adrenal gland glandular cells); on the kidney slide, adjacent cells outside glomeruli should show background staining only if independently confirmed as RBMS3-negative, because the supplied HPA rows do not classify them.
Positive control tissue: Breast (Glandular cells, HPA High)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RBMS3 in A-431, U-251MG, U2OS, with annotated localisation: Vesicles (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit isotype control, and RBMS3 knockout tissue or cells as a biological negative (caption: rabbit anti-RBMS3 antibody). Quench endogenous peroxidase and check endogenous biotin background, especially in renal tubules, when using the caption’s biotin-based DAB detection (caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the fixative in the selected A12416-3 paraffin-section caption is unreported (selected-SKU caption: fixative not stated). The reported IHC condition uses heat retrieval in citrate buffer at pH 6 for 20 minutes, but retrieval dependence has not been established (selected-SKU caption: citrate retrieval); frozen sections are not documented, and IF/ICC is documented through HPA images without evidence that it is easier than IHC (HPA: A-431, U-251MG and U2OS ICC-IF images). In kidney, endogenous biotin in renal tubules can complicate interpretation with biotin-based detection (selected-SKU caption: SABC detection).
HPA tissue IHC evidence for RBMS3
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 |
|---|---|---|---|---|
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in glomeruli | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RBMS3 IHC Tips
Use the catalog antibody’s paraffin-section protocol as a starting point, then judge RBMS3 staining by compartment, cell type, controls, and reproducibility.
What retrieval should I start with for RBMS3 in paraffin sections?
Start with heat-mediated citrate retrieval at pH 6 (datasheet A12416-3). The selected tissue image used 20 minutes of citrate retrieval before staining a paraffin-embedded section (caption A12416-3). Keep section thickness, heating, and cooling consistent across the test and control slides so retrieval differences do not masquerade as expression differences (standard IHC practice). If staining is weak, compare a small retrieval time series before changing buffer chemistry, and inspect tissue structure for heat damage (standard IHC practice). Record the exact retrieval conditions alongside the cytoplasmic staining pattern expected from the tissue profile (HPA: cytoplasmic expression in several tissues).
How should I troubleshoot fixation-related loss of RBMS3 staining?
Target-specific sensitivity to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (caption A12416-3). Record the fixative, fixation duration, processing history, and section age for each specimen before attributing a weak result to RBMS3 biology (standard IHC practice). Compare specimens processed alike and include a concurrently stained tissue control, since fixation and processing can change epitope accessibility in paraffin IHC (standard IHC practice). Adjust retrieval or antibody concentration one variable at a time, while checking tissue preservation and background (standard IHC practice). Do not infer fixation tolerance from the reported tissue staining pattern (HPA: tissue IHC profile).
Should I score diffuse cytoplasmic or punctate RBMS3 staining?
Assess cytoplasmic staining first, because RBMS3 is annotated in the cytoplasm and the tissue IHC profile reports cytoplasmic expression (UniProt Q6XE24: subcellular location; HPA: tissue IHC profile). Vesicle-like puncta can also be considered, since the subcellular IF record lists vesicles as its approved location (HPA: subcellular summary). Score diffuse and punctate patterns separately rather than treating every dark deposit as equivalent (standard IHC practice). Compare each pattern with the no-primary control, tissue morphology, and its distribution across intact cells (standard IHC practice). Treat isolated nuclear or extracellular staining cautiously because those compartments lack support in the supplied localisation records (UniProt Q6XE24; HPA: subcellular summary).
Can this antibody distinguish RBMS3 isoforms in paraffin sections?
Do not assign an IHC signal to a particular isoform without an antibody epitope map and isoform-specific validation (standard IHC interpretation). RBMS3 has 5 listed isoforms, and the canonical sequence contains RRM 1 at residues 61–134 and RRM 2 at 140–225 (UniProt Q6XE24). The supplied product caption documents tissue staining but gives no epitope or isoform selectivity (caption A12416-3). Ask whether a candidate epitope is present in the isoforms under study before comparing stained compartments or specimens (standard antibody validation practice). Report the result as RBMS3 immunoreactivity unless independent evidence establishes which isoform the antibody detects (standard IHC interpretation).
How can I check an RBMS3 IHC pattern by multiplex IF?
Use IF as an orthogonal check of the chromogenic pattern, while validating the antibody and controls for IF separately (standard immunostaining practice). Pair RBMS3 with a marker of the cell type being assessed, such as a glandular epithelial marker when examining the glandular-cell signal reported in breast tissue (HPA: high in breast glandular cells). Choose a far-red fluorophore where tissue autofluorescence obscures shorter-wavelength channels, and include single-color and no-primary controls (standard IF practice). After fixation, use and titrate permeabilisation sufficient to reach a cytoplasmic epitope; RBMS3 has no annotated transmembrane segment (UniProt Q6XE24: topology and localisation). Compare puncta separately because the IF record places RBMS3 in vesicles (HPA: subcellular summary).
What should I change when RBMS3 DAB staining looks diffuse or granular?
Start by examining a no-primary section, then compare the suspected signal with intact-cell cytoplasm and vesicle-like structures (standard IHC practice; UniProt Q6XE24: cytoplasm; HPA: subcellular summary). The selected paraffin-section image used 10% goat serum, 1 µg/mL primary antibody overnight at 4°C, and biotin-based DAB detection (caption A12416-3). If background is widespread, titrate the primary concentration and review washing, blocking, and secondary-only controls before changing retrieval (standard IHC practice). Include a peroxidase block for DAB work and assess whether the biotin-based detection system contributes background in the specimen (standard chromogenic IHC practice). Distinguish granular deposits on damaged edges from staining enclosed by preserved cell boundaries (standard IHC practice).
How should I quantify RBMS3 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic compartment before scoring, consistent with the reported RBMS3 tissue pattern (HPA: cytoplasmic expression in several tissues). For cell-based comparison, record the percentage of positive cells and intensity categories, then calculate an H-score using the same thresholds across slides (standard IHC scoring practice). Alternatively, measure positive-cell density per mm² of viable tissue when cell counts better fit the specimen architecture (standard image-analysis practice). Normalise to the number of eligible cells or viable tissue area, and document exclusions for folds, edges, and necrosis (standard IHC scoring practice). Keep retrieval, imaging, and threshold settings consistent, and report controls with each batch (standard IHC practice).
How do I separate genuine RBMS3 staining from artefact?
Give greatest weight to reproducible staining within intact-cell cytoplasm, while reviewing vesicle-like puncta separately (UniProt Q6XE24: cytoplasm; HPA: subcellular summary). Check that the stained cell type matches the claim: the tissue profile reports high staining in breast glandular cells but no detection in esophageal squamous epithelial cells (HPA: tissue IHC profile). The selected antibody image depicts staining in a paraffin-embedded human oesophagus squama cancer section, so it does not establish staining in normal esophageal epithelium (caption A12416-3). Exclude edge effects, necrosis, and endogenous peroxidase with morphology and appropriate controls (standard IHC practice). Interpret apparent positives cautiously because tissue IHC reliability is rated uncertain owing to low agreement with RNA data (HPA: reliability).
Boster reagents
Best RBMS3 / RNA-binding motif, single-stranded-interacting protein 3 IHC Antibodies
A12416-3 has IHC images from human oesophagus squama cancer and mouse and rat brain paraffin sections, plus an IF image from U20S cells (catalog image captions).
Real IHC data
Anti-RBMS3 Antibody ®
Cat # A12416-3
A12416-3 has IHC images from human oesophagus squama cancer and mouse and rat brain paraffin sections (catalog IHC captions). Its IF image shows U20S cells, and its listed applications include IF and ICC (catalog IF caption; catalog applications).
Which to pick: Choose A12416-3 for paraffin-section IHC; its own captions document staining in human oesophagus squama cancer and mouse and rat brain, while the fixative is unreported (catalog IHC captions). Choose A12416-3 for IF/ICC based on its listed applications and IF image of U20S cells (catalog applications; catalog IF caption). It is listed as reactive with Human, Mouse, and Rat; clonality is unreported (catalog reactivity; catalog clone field).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q6XE24 (RBMS3_HUMAN, RNA-binding motif, single-stranded-interacting protein 3).
- Human Protein Atlas. RBMS3 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. RBMS3 subcellular location (ICC-IF): Localized to vesicles..
- Human Protein Atlas. RBMS3 antibody validation summary (1 antibodies).
- Expression of RBMS3 in Breast Cancer Progression. International journal of molecular sciences 2023 — PMC9917836.
- Increased expression of RBMS3 predicts a favorable prognosis in human gallbladder carcinoma. Oncology reports 2020 — PMC7251710.
- Down regulation of RNA binding motif, single-stranded interacting protein 3, along with up regulation of nuclear HIF1A correlates with poor prognosis in patients with gastric cancer. Oncotarget 2017 — PMC5352053.
- Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration. Heliyon 2024 — PMC11079397.
- PubMed PMID:10675610 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16641997 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RBMS3 (RNA-binding motif, single-stranded-interacting protein 3). Expected localisation Cytoplasmic staining in several tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A12416-3). Validated IHC antibody: A12416-3 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Breast (Glandular cells); negative Adrenal gland. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human oesophagus squama cancer tissues; fixative not specified (datasheet A12416-3); 2. Fixation: Image fixative and duration unreported (datasheet A12416-3); 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: Citrate pH 6 , 20 min (datasheet A12416-3); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A12416-3); 8. Primary antibody: Rabbit anti-RBMS3, 0.5-1μg/ml (datasheet A12416-3); 9. Primary incubation: Overnight at 4 °C (datasheet A12416-3). Watch for: Fixation: Keep fixation consistent across compared paraffin sections. (selected-SKU IHC image A12416-3); Caveat: Tissue staining has low concordance with RNA data (HPA tissue IHC); Isoform / epitope: 5 isoforms; epitope coverage needs checking (UniProt); Regulation: Low tissue specificity at RNA level (HPA tissue RNA). Evidence: UniProt Q6XE24, HPA tissue IHC · Uncertain, HPA ICC-IF · vesicles., Datasheet A12416-3, PMC9917836, PMC7251710. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RBMS3/immunohistochemistry-ihc-guide.