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RPS9 / Small ribosomal subunit protein uS4 · IHC design guide
Design Immunohistochemistry for RPS9
Plan RPS9 chromogenic IHC in paraffin sections using the cytoplasmic tissue pattern reported by HPA (HPA tissue IHC). This guide covers fixation, staining interpretation and controls, including marrow peroxidase background (standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RPS9 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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic in glandular and hematopoietic cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 8.0 HIER, heat-mediated (datasheet A05633-1) | |
| Positive control | Bone marrow+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 consistent across paraffin sections (standard IHC practice; not target-specific) | |
| Caveat | Endogenous peroxidase in marrow may raise background (standard IHC practice) | |
| Regulation | Expression regulation unannotated (UniProt) | |
| Isoform / epitope | No annotated isoforms; chain spans residues 2–194 (UniProt) |
Section 1
Recommended RPS9 IHC & IF Protocols
The catalog antibody uses heat-mediated Tris-EDTA retrieval at pH 8.0 (datasheet A05633-1). One published RPS9 IHC protocol follows (PMC5604204).
| Sample | Paraffin-embedded Human brain tissue; fixative not specified (datasheet A05633-1) |
| Fixation | Image fixative and duration unreported (datasheet A05633-1); 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 8.0 (datasheet A05633-1); 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-RPS9, 1:100 - 1:300 (datasheet A05633-1) |
| 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 | RPS9-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA at pH 8.0 (datasheet A05633-1); consider the published EDTA pH 9.0 method when optimizing staining (PMC5604204).
Section 2
What Is the Expected RPS9 Staining Pattern?
RPS9 is a small ribosomal subunit protein reported in the cytoplasm and nucleolus, with no transmembrane segment (UniProt P46781: function, location, topology). For paraffin section IHC, expect predominantly cytoplasmic staining in several tissues, including strong staining in selected glandular and hematopoietic cells (HPA: tissue IHC). HPA rates its tissue IHC profile Approved, with medium consistency between antibody staining and RNA expression (HPA: tissue IHC reliability).
| Clear cytoplasmic staining in pancreatic exocrine glandular cells or bone marrow hematopoietic cells. | These are suitable positive patterns: both cell populations are rated High by HPA (HPA: pancreas and bone marrow tissue IHC). Judge the signal in the named cells, since a tissue section contains multiple cell types (standard IHC practice). |
| A nucleolar component accompanies otherwise plausible cytoplasmic staining. | Nucleolar localization is compatible with RPS9 biology (UniProt P46781: subcellular location). HPA describes the tissue IHC profile as cytoplasmic, so a prominent nuclear pattern alone needs independent confirmation before being called RPS9 specific (HPA: tissue IHC profile; standard IHC practice). |
| Staining is confined to cell borders or extracellular material, with little cytoplasmic signal. | That distribution conflicts with the reported cytoplasmic and nucleolar locations and absence of a transmembrane segment (UniProt P46781: location, topology). Treat it as a suspect IHC pattern and check the detection controls before assigning it to RPS9 (standard IHC practice). |
| Strong staining appears in cardiomyocytes or adipocytes, while expected positive cells are weak. | HPA reports RPS9 as Not detected in cardiomyocytes and adipocytes (HPA: heart muscle and adipose tissue IHC). The mismatch can suggest cross-reactivity or endogenous detection activity; it does not establish either cause from one slide (standard IHC practice). |
| Diffuse color covers many compartments, or no signal appears in a known positive cell population. | Diffuse color prevents a confident compartment call (standard IHC practice). Absent staining in a High HPA population, such as duodenal glandular cells, leaves the run uninterpretable until controls and tissue preservation are reviewed (HPA: duodenum tissue IHC; standard IHC practice). |
💡Expected RPS9 appearanceCall a result positive when named HPA High cells show convincing cytoplasmic staining, with a possible nucleolar component; isolated border or extracellular color is suspect (HPA: tissue IHC; UniProt P46781: location, topology).
| Cell and tissue choice | Use a named High population, such as small intestinal glandular cells, to assess the run (HPA: small intestine tissue IHC). HPA reports low tissue RNA specificity, so an unlisted tissue is not automatically negative (HPA: tissue RNA specificity). |
| Intracellular location | RPS9 is reported in cytoplasm and nucleolus, including cytoplasmic mRNP granules (UniProt P46781: subcellular location). HPA's observed tissue IHC profile is cytoplasmic; score what the stained cells show, without requiring visible granules (HPA: tissue IHC profile; standard IHC practice). |
| Antibody validation | The listed antibody, HPA048746, has Approved IHC status; no ICC status is supplied (HPA: antibody validation). HPA's tissue reliability description is medium consistency with RNA expression, so validate an unexpected pattern independently (HPA: tissue IHC reliability; standard IHC practice). |
| Sequence and epitope interpretation | UniProt reports one processed chain spanning residues 2–194, no signal peptide or propeptide, and no listed isoforms (UniProt P46781: processing, isoforms). It lists modified residues, but the supplied sources do not locate this antibody's epitope or establish a staining effect (UniProt P46781: modified residues; HPA: antibody record). |
| IF/ICC Q&A | What pattern should IF/ICC show? Cytoplasmic or nucleolar signal is biologically plausible (UniProt P46781: location), but HPA supplies no main ICC location, image bearing cell line, or ICC validation for the listed antibody; treat an IF result as unverified here (HPA: subcellular and antibody records). |
| Situation | Likely cause | Next action |
|---|---|---|
| High background obscures cell boundaries. | Nonspecific reagent binding, endogenous detection activity, or excess detection signal are possible general IHC causes (standard IHC practice). | Inspect a no primary control, adjust blocking or detection conditions, and compare with a named HPA High population before scoring RPS9 (standard IHC practice; HPA: tissue IHC). |
| No staining in pancreatic exocrine glandular cells. | A failed run, unsuitable reagent conditions, or poor tissue preservation are possibilities; HPA rates these cells High (standard IHC practice; HPA: pancreas tissue IHC). | Verify section quality, primary antibody application, retrieval and detection controls, then repeat alongside a positive section (standard IHC practice). No RPS9 specific retrieval condition is supplied (HPA: supplied record). |
| Only nuclear staining is apparent. | UniProt permits nucleolar RPS9, while HPA describes tissue staining as cytoplasmic; the observed pattern needs closer compartment assessment (UniProt P46781: location; HPA: tissue IHC profile). | Examine nuclear detail against the counterstain and compare with cytoplasmic signal in an HPA High cell population; seek independent confirmation if the pattern remains nuclear only (standard IHC practice; HPA: tissue IHC). |
| Cardiomyocytes stain strongly in a heart section. | HPA reports RPS9 as Not detected in cardiomyocytes; cross-reactivity or endogenous detection activity is possible, but neither is proven (HPA: heart muscle tissue IHC; standard IHC practice). | Check a no primary control and repeat with an appropriate independent control before interpreting the heart signal as RPS9 (standard IHC practice). |
| A visually clean negative section is used as the only run control. | A negative section cannot demonstrate that the primary and detection steps produced a detectable RPS9 signal (standard IHC practice). | Include a named HPA High cell population and score that population directly; HPA lists adipocytes and smooth muscle cells as Not detected comparators (HPA: tissue IHC). |
| An IF/ICC image is being used to settle an ambiguous IHC result. | HPA supplies no ICC image bearing cell line, main ICC location, or ICC validation status for HPA048746 (HPA: subcellular and antibody records). | Interpret the paraffin section against its own controls and HPA tissue IHC pattern; regard any IF/ICC observation as a separate, unvalidated line of evidence here (HPA: tissue IHC, subcellular and antibody records; standard IHC practice). |
Experiment evidence
IHC & IF images showing RPS9 staining
A05633-1 · IHCImmunohistochemical analysis of paraffin-embedded Human brain. Antibody was diluted at 1:100(4° overnight). High-pressure and temperature Tr
A05633-1 · IFImmunofluorescence analysis of A549 cells, using RPS9 Antibody. The picture on the right is blocked with the synthesized peptide.
A05633S9 · IHCImmunohistochemical analysis of paraffin-embedded Human brain. Antibody was diluted at 1:100 (4° overnight). High-pressure and temperature T
A05633S9 · IFImmunofluorescence analysis of A549 cells, using RPS9 Antibody. The picture on the right is blocked with the synthesized peptide.
A05633 · IHCImmunohistochemistry (IHC) analyzes of RPS9 (E73) pAb in paraffin-embedded human colorectal carcinoma tissue at 1:50.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000170889-RPS9/tissue/cerebral+cortex#imid_13472739
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000170889-RPS9/tissue/colon#imid_13461153
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000170889-RPS9/tissue/liver#imid_13461138
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000170889-RPS9/tissue/kidney#imid_13461147
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RPS9 IHC & IF
🧪Run bone marrow first; hematopoietic cells should stain (HPA: High in bone marrow hematopoietic cells), while any adipocytes on the same slide should show background-level staining as provisional internal comparators (HPA: Not detected in adipose-tissue adipocytes). Run adipose tissue as the negative comparator, assessing its adipocytes for background-level staining (HPA: Not detected in adipocytes).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for RPS9; derive a cell-line control from the positive tissue's cell type (Hematopoietic cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) control, a control matched to the primary antibody’s host species and isotype, and an immunogen-peptide preabsorption control (selected-SKU tissue-IHC caption: peptide-preabsorbed negative control). Quench endogenous peroxidase in bone marrow before chromogenic detection and check for residual background (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A05633-1 paraffin-brain caption does not state a fixative (selected-SKU tissue-IHC caption). That caption reports high-pressure, high-temperature Tris-EDTA retrieval at pH 8.0 and antibody incubation at 1:100 overnight at 4 °C; whether retrieval is required for RPS9 is unreported (selected-SKU tissue-IHC caption). The supplied evidence does not establish frozen sections or IF/ICC as easier, and endogenous peroxidase may cause background in bone marrow chromogenic IHC (standard IHC practice).
HPA tissue IHC evidence for RPS9
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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Pancreas | Exocrine glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Nasopharynx | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Follicle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RPS9 IHC Tips
Troubleshoot RPS9 staining in paraffin section IHC using the catalog antibody’s tissue image, HPA tissue patterns, and known cellular localisation.
Which retrieval conditions should I start with for weak RPS9 staining?
Start with heat mediated Tris-EDTA retrieval at pH 8.0 for paraffin sections (datasheet A05633-1). The selected human brain image used high pressure and temperature retrieval, followed by antibody at 1:100 overnight at 4°C (caption A05633-1). If staining is weak, compare retrieval duration across adjacent sections while holding dilution, detection, and development constant (standard IHC practice). Use a section with readily detectable glandular or hematopoietic cells as a process control, and inspect morphology for heat damage before increasing retrieval intensity (HPA: high staining in several glandular populations and bone marrow hematopoietic cells; standard IHC practice).
Could fixation explain weak or uneven RPS9 staining?
Target specific fixation sensitivity is unknown: the selected caption identifies paraffin embedded human brain but does not report its fixative (caption A05633-1). Record the actual fixative and fixation time for each specimen, then compare adjacent sections processed with the same Tris-EDTA retrieval at pH 8.0 (datasheet A05633-1; standard IHC practice). Uneven staining that follows folds, thick areas, or damaged edges warrants a section quality check before changing antibody conditions (standard IHC practice). Keep antibody dilution and overnight incubation consistent during that comparison, using 1:100 at 4°C as the documented image conditions (caption A05633-1).
Where should convincing RPS9 chromogen appear within cells?
Expect predominantly cytoplasmic staining in tissue sections, consistent with the reported IHC profile across several tissues (HPA: cytoplasmic expression in several tissues). Nuclear and nucleolar localisation is biologically plausible, and RPS9 can also occur in cytoplasmic mRNP granules (UniProt P46781 subcellular annotation). Assess these compartments within intact cells against a nuclear counterstain, rather than scoring diffuse chromogen outside cell boundaries (UniProt P46781 subcellular annotation; standard IHC practice). A sharply membranous pattern would be unexpected because RPS9 has no transmembrane segment; investigate it with a no primary control and nearby tissue morphology (UniProt P46781 topology; standard IHC practice).
How should I troubleshoot compartment dependent or patchy epitope staining?
RPS9 has no annotated isoforms, so a compartment difference alone does not establish isoform selective recognition (UniProt P46781 isoforms and subcellular annotation). Its S4 RNA binding domain spans residues 108–182, while reported modifications include acetyllysines at 66, 116, and 155 and phosphoserines at 153 and 163 (UniProt P46781 domains and modified residues). Without a mapped antibody epitope, these features cannot identify the cause of patchy staining; compare retrieval and section quality under otherwise matched conditions (datasheet A05633-1; standard IHC practice). The selected image includes an immunogen peptide preabsorption control, which offers a relevant comparison for staining attributed to this antibody (caption A05633-1).
How can IF help investigate an ambiguous RPS9 IHC pattern?
Use IF as a separate validation experiment, since the supplied HPA subcellular record lists no ICC/IF image bearing cell lines (HPA: subcellular record). Multiplex RPS9 with a marker for the expected cell population, choosing a fluorophore channel away from prominent tissue autofluorescence and checking single stain controls (HPA: tissue cell patterns; standard IF practice). RPS9 has no transmembrane segment and is annotated in cytoplasm, nucleus, and nucleolus, so permeabilisation should permit access to intracellular epitopes (UniProt P46781 topology and subcellular annotation; standard IF practice). Compare compartment patterns with chromogenic IHC cautiously because the selected antibody image documents paraffin section IHC conditions, not IF performance (caption A05633-1).
How do I distinguish RPS9 staining from chromogenic background?
Include a no primary control to assess detection background and compare it with the immunogen peptide preabsorption control shown for the selected antibody (caption A05633-1; standard IHC practice). For peroxidase detection, block endogenous peroxidase before DAB development and assess pigment or precipitate in the counterstained section (standard IHC practice). Reduce excessive chromogen development or antibody concentration only after checking whether staining follows intact cell boundaries and the expected cytoplasmic distribution (HPA: cytoplasmic tissue profile; standard IHC practice). Treat staining at cut edges, folds, or necrotic areas as suspect until it is reproducible in preserved tissue and absent from the relevant controls (standard IHC practice).
What scoring method makes RPS9 IHC comparisons defensible? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic scoring compartment before measuring sections, because HPA reports cytoplasmic staining with differences among cell types (HPA: tissue IHC profile). For intact cells, report the percentage positive and an H-score based on intensity categories; where discrete positive cells matter, report density per mm² of evaluable tissue (standard IHC practice). Normalise counts to the relevant cell population or intact tissue area, and apply one threshold and chromogen development rule across the comparison (standard IHC practice). Exclude folds and necrosis, retain the no primary background control, and report how many fields and specimens contributed to each score (standard IHC practice).
When should an apparent RPS9 positive cell be considered artefactual?
Give greatest weight to staining in intact cytoplasm of the expected cell type; HPA reports high staining in bone marrow hematopoietic cells and several glandular populations (HPA: tissue IHC profile). Interpret signal cautiously in cell types reported as undetected, such as adipocytes or cardiomyocytes, while recognising the tissue antibody record has medium consistency with RNA expression (HPA: tissue IHC profile and reliability description). A purely membranous pattern conflicts with the absence of a transmembrane segment, whereas nuclear or nucleolar signal remains plausible (UniProt P46781 topology and subcellular annotation). Reject edge and necrotic deposits or endogenous enzyme signal when they appear in controls or lack preserved cellular morphology (standard IHC practice).
Boster reagents
Best RPS9 / Small ribosomal subunit protein uS4 IHC Antibodies
Three anti-RPS9 antibodies have IHC images from paraffin-embedded human brain or colorectal carcinoma (catalog image captions); two also have IF images from A549 cells (catalog IF captions). All list Human, Mouse and Rat reactivity (catalog).
Real IHC data
Anti-40S ribosomal protein S9 RPS9 Antibody
Cat # A05633-1
Real IHC data
Anti-Ribosomal Protein S9 RPS9 Antibody
Cat # A05633S9
Real IHC data
Anti-RPS9 (E73) Antibody
Cat # A05633
A05633-1 and A05633S9 each show IHC in paraffin-embedded human brain and IF in A549 cells (respective catalog image captions); A05633-1 also lists ICC (catalog applications). A05633 shows IHC in paraffin-embedded human colorectal carcinoma at 1:50 (A05633 image caption); all three list Human, Mouse and Rat reactivity (catalog).
Which to pick: For tissue IHC, choose by the sample shown: A05633-1 or A05633S9 for paraffin-embedded human brain, each shown at 1:100 with Tris-EDTA pH 8.0 retrieval (respective IHC captions), or A05633 for paraffin-embedded human colorectal carcinoma (A05633 IHC caption). For IF, A05633-1 and A05633S9 have A549 images; choose A05633-1 if ICC is required because ICC appears in its application list (catalog IF captions; catalog applications). For other species, all three list Mouse and Rat reactivity and are rabbit polyclonals (catalog), but the supplied IHC images show human specimens only (respective IHC captions); the fixative is unreported in those captions.
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P46781 (RS9_HUMAN, Small ribosomal subunit protein uS4).
- Human Protein Atlas. RPS9 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. RPS9 subcellular location (ICC-IF): Variation in transcript expression is correlated to the cell cycle..
- Human Protein Atlas. RPS9 antibody validation summary (1 antibodies).
- Down-regulation of RPS9 Inhibits Osteosarcoma Cell Growth through Inactivation of MAPK Signaling Pathway. Journal of Cancer 2017 — PMC5604204.
- PubMed PMID:7772601 — UniProt-cited evidence.
- PubMed PMID:11875025 — UniProt-cited evidence.
- PubMed PMID:15057824 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RPS9 (Small ribosomal subunit protein uS4). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 8.0 HIER, heat-mediated (datasheet A05633-1). Validated IHC antibody: A05633-1 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded Human brain tissue; fixative not specified (datasheet A05633-1); 2. Fixation: Image fixative and duration unreported (datasheet A05633-1); 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 retrieval: Tris-EDTA pH 8.0 (datasheet A05633-1); 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-RPS9, 1:100 - 1:300 (datasheet A05633-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Endogenous peroxidase in marrow may raise background (standard IHC practice); Isoform / epitope: No annotated isoforms; chain spans residues 2–194 (UniProt); Regulation: Expression regulation unannotated (UniProt). Evidence: UniProt P46781, HPA tissue IHC · Approved, HPA cell-line RNA · Variation in transcript expression is correlated to the cell cycle., Datasheet A05633-1, PMC5604204. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RPS9/immunohistochemistry-ihc-guide.