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RPS28 / Small ribosomal subunit protein eS28 · IHC design guide
Design Immunohistochemistry for RPS28
Plan chromogenic RPS28 IHC in paraffin sections using the cytoplasmic tissue pattern (HPA tissue IHC). Duodenal glandular cells show high staining, while adipocytes are not detected (HPA tissue IHC); the catalog antibody's IHC dilution is 2–5 μg/mL (datasheet A10672).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RPS28 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 staining in neuronal, glandular and squamous cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A10672) | |
| Positive control | Caudate+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA expression show medium consistency (HPA tissue IHC) | |
| Regulation | No specific expression regulator annotated (UniProt) | |
| Isoform / epitope | No annotated isoforms; one 1–69 chain (UniProt) |
Section 1
Recommended RPS28 IHC & IF Protocols
The catalog antibody protocol uses EDTA pH 8.0 retrieval (datasheet A10672). Three published IHC protocols provide tissue-specific staining conditions (PMC9117278; PMC10776981; PMC13578613).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A10672) |
| Fixation | Image fixative and duration unreported (datasheet A10672); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A10672); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A10672) |
| Primary antibody | Rabbit anti-RPS28, 2-5μg/ml (datasheet A10672) |
| Primary incubation | Overnight at 4 °C (datasheet A10672) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A10672) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RPS28-positive staining in neuronal cells of caudate (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet A10672); optimize retrieval for other antibodies when the article does not specify it.
Section 2
What Is the Expected RPS28 Staining Pattern?
RPS28 is a 40S ribosomal protein expected mainly in cytoplasm, including cytosol and rough endoplasmic reticulum; UniProt also lists the nucleolus (UniProt P62857). In tissue IHC, look for cytoplasmic staining in the cell types documented below (HPA tissue IHC). RPS28 has no transmembrane segment (UniProt P62857). HPA rates the tissue staining Approved, with medium consistency between antibody staining and RNA expression (HPA tissue IHC).
| Cytoplasmic staining in duodenal glandular cells or pancreatic exocrine glandular cells. | This matches HPA's High staining in those cell types and its overall cytoplasmic tissue profile (HPA tissue IHC). Score the specified cells, rather than treating the whole tissue as uniformly positive. |
| Predominantly nuclear or membrane-edge staining, with little cytoplasmic signal. | Recheck the result against HPA's cytoplasmic tissue pattern and UniProt's lack of a transmembrane segment (HPA tissue IHC; UniProt P62857). Nucleolar localization is listed by UniProt, so a nucleolar component alone does not establish an artefact (UniProt P62857). |
| Strong staining in adipocytes or cardiomyocytes. | HPA reports RPS28 as Not detected in those cells (HPA tissue IHC). Consider cross-reactivity or endogenous detection activity; compare a documented positive tissue and a control omitting the primary antibody before assigning the signal to RPS28. |
| Diffuse color across cells and surrounding tissue, without a clear cytoplasmic pattern. | The distribution is difficult to reconcile with HPA's cytoplasmic profile (HPA tissue IHC). Check a control omitting the primary antibody and inspect blocking, washes and detection conditions as general IHC troubleshooting steps. |
| No signal in duodenal glandular cells despite a visible counterstain. | HPA reports High staining in those cells (HPA tissue IHC). First confirm that the expected cells are present; then review the antibody's IHC validation and the assay's retrieval and detection steps. A failed positive control cannot establish true absence. |
💡Expected RPS28 appearanceCall a result consistent with RPS28 when the expected cells show chiefly cytoplasmic staining, potentially High in duodenal glandular or pancreatic exocrine cells; isolated membrane-edge or widespread diffuse color is suspect (HPA tissue IHC; UniProt P62857 topology).
| Cell type and tissue (HPA tissue IHC) | HPA reports High staining in several neuronal, glandular and squamous epithelial cell types, but Not detected in adipocytes, cardiomyocytes and others (HPA tissue IHC). Choose and score controls at the cell-type level; a tissue name alone is insufficient. |
| Subcellular distribution (UniProt P62857; HPA tissue IHC) | Cytosol and rough endoplasmic reticulum are listed by UniProt, while HPA summarizes tissue staining as cytoplasmic (UniProt P62857; HPA tissue IHC). UniProt also lists the nucleolus; interpret any nuclear component alongside the dominant tissue pattern. |
| Antibody evidence (HPA antibodies; HPA tissue IHC) | HPA047132 is Approved for IHC, and the tissue profile has medium consistency with RNA expression (HPA antibodies; HPA tissue IHC). These ratings support use of the reference pattern but do not make unexpected staining proof of RPS28. |
| IF/ICC Q: What localization should I expect? | A: HPA reports approved cytosolic localization and lists A-431 and U2OS images; HPA047132 is Approved for ICC (HPA subcellular; HPA antibodies). Use the separate IF/ICC guide for that application. |
| Antigen retrieval and fixation | Target-specific fixation sensitivity and retrieval effects are unreported in the supplied UniProt and HPA records. Follow the IHC-validated antibody's documented workflow, and interpret retrieval changes as assay checks rather than established RPS28 behavior. |
| Situation | Likely cause | Next action |
|---|---|---|
| Documented positive cells are blank. | The cells may be absent from the section, or the IHC assay may have failed; duodenal glandular cells are High in HPA (HPA tissue IHC). | Confirm cell identity and run a documented positive tissue alongside the sample. Check antibody, retrieval, detection and counterstain steps using the chosen IHC workflow. |
| Only nuclei stain strongly. | A nuclear-only result differs from HPA's cytoplasmic tissue profile, although UniProt lists nucleolar localization (HPA tissue IHC; UniProt P62857). | Inspect whether signal is nucleolar and whether cytoplasm also stains. Repeat with a documented positive tissue and controls before calling the nuclear signal specific. |
| Adipocytes or cardiomyocytes stain strongly. | Both are Not detected in HPA tissue IHC; nonspecific binding or endogenous detection activity is possible (HPA tissue IHC). | Compare the suspect cells with a positive control and a control omitting the primary antibody. Review blocking and detection conditions if color persists without primary antibody. |
| Color is diffuse across the section. | A uniform haze obscures the cytoplasmic pattern described by HPA (HPA tissue IHC); excess background can arise during general IHC detection. | Inspect the control omitting the primary antibody. Review washes, blocking and detection exposure, then reassess whether individual expected cells retain cytoplasmic staining. |
| A presumed negative tissue shows a few positive cells. | HPA levels apply to named cell types, and its tissue IHC profile has medium consistency with RNA expression (HPA tissue IHC). | Identify the stained cells before scoring. Compare their compartment and distribution with a documented positive; do not label an entire section negative from its tissue name. |
| IF/ICC localization differs from the IHC section. | HPA reports approved cytosolic ICC-IF localization and a cytoplasmic tissue IHC profile (HPA subcellular; HPA tissue IHC). | Score each application against its own HPA reference and controls. Use the separate IF/ICC guide for imaging and assay setup. |
Experiment evidence
IHC & IF images showing RPS28 staining
A10672 · IHCIHC analysis of RPS28 using anti-RPS28 antibody (A10672). RPS28 was detected in a paraffin-embedded section of human colon cancer tissue. He
A10672 · IHCIHC analysis of RPS28 using anti-RPS28 antibody (A10672). RPS28 was detected in a paraffin-embedded section of human endometrial cancer tiss
A10672 · IFIF analysis of RPS28 using anti-RPS28 antibody (A10672) and anti-Tubulin Alpha antibody (M03989-3). RPS28 was detected in immunocytochemical
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000233927-RPS28/tissue/cerebral+cortex#imid_12927671
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000233927-RPS28/tissue/colon#imid_12927597
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000233927-RPS28/tissue/liver#imid_12927598
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000233927-RPS28/tissue/kidney#imid_12927599
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA047132; green = RPS28, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA047132; green = RPS28, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RPS28 IHC & IF
🧪Run caudate first; neuronal cells should stain (HPA: High in caudate neuronal cells). Use adipose tissue as the negative comparison (HPA: Not detected in adipocytes); acellular spaces within the caudate section should remain unstained, while other cell types should not be assumed negative without validation.
Positive control tissue: Caudate (Neuronal cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RPS28 in A-431, U2OS, with annotated localisation: Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a host- and clonality-matched rabbit IgG control, and an RPS28 knockout sample or validated peptide-block control if available (A10672 tissue-IHC caption: rabbit primary antibody). Quench endogenous peroxidase for HRP/DAB detection (A10672 tissue-IHC caption: HRP/DAB); for brain IF, assess lipofuscin autofluorescence with an unstained section (standard IF practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the fixative for the A10672 paraffin section is unreported (A10672 tissue-IHC caption). Heat-mediated retrieval in EDTA at pH 8.0 was used, but the caption does not establish that retrieval is required (A10672 tissue-IHC caption). Is IF/ICC easier? HPA documents cytosolic ICC-IF localization in A-431 and U2OS cells, but the supplied evidence does not establish whether frozen sections or IF are easier than paraffin IHC; brain lipofuscin can complicate IF interpretation (HPA: Cytosol approved; standard IF practice).
HPA tissue IHC evidence for RPS28
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | High | 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 → |
| Ovary | Follicle cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | Chondrocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RPS28 IHC Tips
Troubleshoot RPS28 chromogenic IHC by checking retrieval, cellular location, controls, and scoring against the documented paraffin-section workflow.
What retrieval conditions should I start with for RPS28 paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for RPS28 paraffin-section IHC (datasheet A10672). The illustrated section used 10% goat-serum blocking and 2 μg/ml primary antibody overnight at 4°C (datasheet A10672). Keep heating and cooling conditions consistent across slides when comparing staining intensity (standard IHC practice). If signal is weak, adjust heating duration on replicate sections before testing another buffer, and check tissue morphology at each setting (standard IHC practice). Look for predominantly cytoplasmic staining rather than judging retrieval by total DAB darkness alone (HPA tissue IHC; standard IHC practice).
Could fixation explain variable RPS28 staining between paraffin-section batches?
RPS28-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state its fixative (datasheet A10672). Record fixative, fixation duration, section thickness, and processing batch before comparing chromogenic intensity between specimens (standard IHC practice). If batches differ, compare matched specimens processed under controlled fixation conditions while keeping retrieval and detection settings constant (standard IHC practice). The documented retrieval uses EDTA at pH 8.0, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet A10672). Assess morphology, compartment-specific staining, and negative controls together; the available evidence does not establish a preferred fixative for RPS28 (datasheet A10672; standard IHC practice).
Should RPS28 stain cytoplasm, nucleoli, or both in chromogenic IHC?
Expect predominantly cytoplasmic staining in tissue sections, consistent with the reported tissue profile (HPA tissue IHC). RPS28 is also annotated in the cytosol, nucleolus, and ribosomes associated with rough endoplasmic reticulum, so nucleolar signal can be biologically plausible (UniProt P62857 localisation). It has no annotated transmembrane segment; a crisp cell-surface outline alone would therefore need additional validation (UniProt P62857 topology; standard IHC practice). Compare signal within identifiable cell populations rather than across whole fields: glandular cells in duodenum are reported high, while adipocytes are not detected (HPA tissue IHC). Confirm unexpected compartments with appropriate staining controls and reproducible morphology (standard IHC practice).
How should I investigate apparent epitope loss or an unusual staining pattern?
The supplied record lists one 1–69 RPS28 chain and no annotated isoforms, so isoform-specific tissue staining is unsupported here (UniProt P62857 processing; isoforms). Its annotated modifications include N-terminal acetylation and phosphorylation at serine 41, but the supplied evidence does not place the catalog antibody’s epitope at either site (UniProt P62857 modified residues; datasheet A10672). Check the antibody’s disclosed immunogen or epitope before attributing a staining difference to either modification (standard IHC practice). Compare matched sections under the documented EDTA pH 8.0 retrieval and consistent detection conditions (datasheet A10672; standard IHC practice). Treat compartment shifts as findings requiring independent confirmation (standard IHC practice).
How can I assess RPS28 localisation by IF alongside this IHC guide?
For the separate IF/ICC application, assess RPS28 against its approved cytosolic location and documented IF images from A-431 and U2OS cells (HPA subcellular). Multiplex with a marker identifying the expected cell population so RPS28 signal can be assigned to the correct cells (standard IF practice). Choose a fluorophore channel after checking tissue autofluorescence and include single-label controls when separating overlapping signals (standard IF practice). Because RPS28 has no transmembrane segment and is annotated in intracellular compartments, permeabilisation should provide access to cytosolic and potential nucleolar epitopes (UniProt P62857 topology; localisation; standard IF practice). Check whether permeabilisation preserves cellular morphology before interpreting compartment differences (standard IF practice).
What should I check when DAB obscures RPS28 cytoplasmic staining?
First inspect a section without primary antibody to identify signal arising from secondary reagents, endogenous peroxidase, or the chromogen (standard IHC practice). Apply a peroxidase-blocking step and keep DAB development consistent across specimens; these are general chromogenic IHC controls (standard IHC practice). The illustrated workflow used 10% goat-serum blocking, 2 μg/ml primary antibody, and a peroxidase-conjugated secondary incubated for 30 minutes at 37°C (datasheet A10672). If diffuse staining persists, compare primary-antibody concentration and wash conditions on matched sections (standard IHC practice). Judge improvement by clearer cellular boundaries and predominantly cytoplasmic signal (HPA tissue IHC; standard IHC practice).
How should I score RPS28 staining across heterogeneous tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define eligible cell populations and viable tissue regions before scoring, then assess cytoplasmic DAB within those cells (HPA tissue IHC; standard IHC practice). Record the percentage of positive cells and intensity categories, or calculate an H-score from intensity-weighted percentages on a 0–300 scale (standard IHC practice). For spatial comparisons, report positive-cell density per mm² of viable, eligible tissue rather than total section area (standard IHC practice). Normalize comparisons to the same cell type, viable area, section-processing batch, and staining run (standard IHC practice). Keep nucleolar and cytoplasmic measurements separate when both are visible, since both locations are annotated for RPS28 (UniProt P62857 localisation; standard IHC practice).
How can I distinguish convincing RPS28 staining from tissue or detection artefacts?
A convincing result follows identifiable cells and is predominantly cytoplasmic, matching the reported tissue pattern (HPA tissue IHC). High staining in duodenal glandular cells is a useful reference; adipocytes are reported as not detected and provide a contrasting cell population (HPA tissue IHC). Nucleolar staining can be plausible, but isolated membrane outlines are harder to reconcile with RPS28’s annotated localisation and lack of a transmembrane segment (UniProt P62857 localisation; topology). Treat section-edge staining, necrotic areas, and signal in the no-primary control as possible artefacts rather than positive cells (standard IHC practice). Check endogenous peroxidase and repeat ambiguous patterns in matched sections before interpreting biological differences (standard IHC practice).
Boster reagents
Best RPS28 / Small ribosomal subunit protein eS28 IHC Antibodies
A10672 has real IHC data from human paraffin sections and IF/ICC data from A549 cells (catalog image captions); its listed reactivity includes human, mouse and rat (catalog reactivity).
Real IHC data
Anti-RPS28 Antibody ®
Cat # A10672
A10672 has IHC images from human colon cancer and endometrial cancer paraffin sections (catalog IHC captions). Its IF/ICC image shows A549 cells, and its listed reactivity is human, mouse and rat (catalog IF caption; catalog reactivity).
Which to pick: Choose A10672 for paraffin-section chromogenic IHC: its human tissue captions report EDTA pH 8 retrieval, 2 μg/ml primary antibody and DAB detection; the fixative is unreported (catalog IHC captions). Choose A10672 for IF/ICC because its A549 image documents 5 μg/ml primary antibody and fluorescent detection (catalog IF caption). For mouse or rat samples, A10672 lists those species as reactive, but the supplied IHC and IF images document human samples only (catalog reactivity; catalog image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P62857 (RS28_HUMAN, Small ribosomal subunit protein eS28).
- Human Protein Atlas. RPS28 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. RPS28 subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. RPS28 antibody validation summary (1 antibodies).
- Comparative proteomic analysis identifies differentially expressed proteins and reveals potential mechanisms of traumatic heterotopic ossification progression. Journal of orthopaedic translation 2022 — PMC9117278.
- Essential genes analysis reveals small ribosomal subunit protein eS28 may be a prognostic factor and potential vulnerability in osteosarcoma. Journal of bone oncology 2024 — PMC10776981.
- Multiomics analyses revealed the roles of the epithelial-mesenchymal transition and novel early candidate diagnostic biomarkers in neonatal necrotising enterocolitis. Clinical and translational medicine 2026 — PMC13578613.
- PubMed PMID:11875025 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:8706699 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RPS28 (Small ribosomal subunit protein eS28). Expected localisation Cytoplasmic staining in several tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A10672). Validated IHC antibody: A10672 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Caudate (Neuronal cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A10672); 2. Fixation: Image fixative and duration unreported (datasheet A10672); 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: EDTA pH 8.0 (datasheet A10672); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A10672); 8. Primary antibody: Rabbit anti-RPS28, 2-5μg/ml (datasheet A10672); 9. Primary incubation: Overnight at 4 °C (datasheet A10672). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA expression show medium consistency (HPA tissue IHC); Isoform / epitope: No annotated isoforms; one 1–69 chain (UniProt); Regulation: No specific expression regulator annotated (UniProt). Evidence: UniProt P62857, HPA tissue IHC · Approved, HPA ICC-IF · cytosol., Datasheet A10672, PMC9117278, PMC10776981. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RPS28/immunohistochemistry-ihc-guide.