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RBBP9 / Serine hydrolase RBBP9 · IHC design guide
Design Immunohistochemistry for RBBP9
Plan RBBP9 staining in paraffin sections using the reported cytoplasmic tissue pattern (HPA tissue IHC). This guide covers fixation, interpretation, and the 2 reported isoforms (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RBBP9 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 most tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in glandular cells and other tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, heat-mediated (datasheet A09378) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A09378) | |
| Caveat | Antibody staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Higher expression in carcinoma tissues (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended RBBP9 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet A09378) with a published RBBP9 paraffin-section IHC workflow (PMC11786897).
| Sample | Paraffin-embedded rat ovary tissue; fixative not specified (datasheet A09378) |
| Fixation | Image fixative and duration unreported (datasheet A09378); 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 6.0 (datasheet A09378); 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-RBBP9, 1:50-1:200 (datasheet A09378) |
| 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 | RBBP9-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 heat retrieval (datasheet A09378). The article also reports EDTA pH 9.0 without specifying which buffer was used for RBBP9 (PMC11786897).
Section 2
What Is the Expected RBBP9 Staining Pattern?
For paraffin-section IHC, expect predominantly cytoplasmic RBBP9 staining in cells such as glandular cells of the colon, duodenum, breast, appendix, and adrenal gland (HPA: tissue IHC). These sites are reported at medium intensity (HPA: tissue IHC). RBBP9 has no annotated transmembrane segment (UniProt O75884: topology). Treat the pattern as provisional: HPA rates tissue IHC “Approved” but reports low consistency between antibody staining and RNA expression (HPA: reliability).
| Cytoplasmic signal in colon or duodenal glandular cells, with a clear cell outline. | This matches the reported compartment and cell types; both are listed at medium staining (HPA: tissue IHC). Compare positive cells with neighboring cells on the same section, then score the proportion and intensity consistently (general IHC practice). |
| Predominantly nuclear staining in a paraffin-section IHC slide. | This differs from the reported cytoplasmic tissue IHC pattern (HPA: tissue IHC). Review the counterstain and detection controls before accepting it as RBBP9; nucleoplasmic localisation belongs to the separate ICC-IF observation (HPA: subcellular ICC-IF). |
| Strong staining in adipocytes, skin fibroblasts, or bone-marrow hematopoietic cells. | Those cell types are listed as not detected (HPA: tissue IHC). Check whether the signal tracks tissue structure or occurs in a no-primary control; cross-reactivity or endogenous detection activity is possible (general IHC practice). |
| Uniform colour across cells, stroma, and section edges. | A field-wide deposit is difficult to reconcile with cell-resolved cytoplasmic staining (HPA: tissue IHC). Review no-primary controls, blocking, washes, and detection exposure for background (general IHC practice). |
| No convincing staining in colon or duodenal glandular cells. | Both are reported at medium staining (HPA: tissue IHC), so first check tissue preservation, retrieval and detection controls (general IHC practice). A failed positive control limits interpretation of other negative areas; HPA’s low RNA–staining consistency also cautions against treating every discrepancy as technical failure (HPA: reliability). |
💡Expected RBBP9 appearanceCall a paraffin-section IHC result consistent when glandular cells show cell-resolved cytoplasmic staining around the reported medium level (HPA: tissue IHC); isolated nuclear-only signal or uniform background needs control review (HPA: tissue IHC; general IHC practice).
| Assay-specific localisation | Tissue IHC is described as cytoplasmic, while ICC-IF has a supported nucleoplasmic main location (HPA: tissue IHC; HPA: subcellular ICC-IF). Interpret each against its own assay evidence; the two observations do not establish the cause of the difference. |
| Antibody evidence | HPA015830 has “Approved” IHC status; HPA049005 has “Supported” ICC status, with no IHC status supplied for that antibody (HPA: antibodies). These labels do not make the two assays interchangeable. |
| Positive and negative comparison cells | Colon and duodenal glandular cells are reported at medium staining; adipocytes and skin fibroblasts are listed as not detected (HPA: tissue IHC). Use the named cell populations, rather than a whole-tissue label, when comparing slides. |
| Topology and isoforms | UniProt lists no transmembrane segment, no signal peptide or propeptide, one chain spanning residues 1–186, and two isoforms (UniProt O75884). Epitope position and isoform coverage are not supplied, so these entries cannot predict antibody staining differences. |
| IF/ICC Q&A | Q: Should IF/ICC show the same compartment as paraffin IHC? A: HPA reports supported nucleoplasmic ICC-IF localisation and cytoplasmic tissue IHC expression (HPA: subcellular ICC-IF; HPA: tissue IHC). Evaluate IF/ICC on its separate guide page. |
| Situation | Likely cause | Next action |
|---|---|---|
| The intended positive glandular cells have little or no signal. | The run may lack usable target detection; the cells are reported at medium staining (HPA: tissue IHC). | Check section quality, retrieval conditions, primary-antibody use and detection reagents against run controls (general IHC practice). Do not infer an RBBP9-specific fixation effect from this result. |
| The slide shows nuclear-only signal. | Its compartment differs from the reported cytoplasmic tissue IHC pattern (HPA: tissue IHC); HPA’s nucleoplasmic observation comes from ICC-IF (HPA: subcellular ICC-IF). | Verify the cellular counterstain, review antibody and detection controls, and report the compartment as a discrepancy if it persists (general IHC practice). |
| Cells listed as not detected stain strongly. | Adipocytes, skin fibroblasts and bone-marrow hematopoietic cells are listed as not detected (HPA: tissue IHC); cross-reactivity or endogenous detection activity may contribute (general IHC practice). | Compare a no-primary control and inspect whether the deposit follows a particular cell type or occurs broadly (general IHC practice). |
| Colour covers the section without clear cellular boundaries. | Diffuse deposition can obscure the reported cytoplasmic pattern (HPA: tissue IHC; general IHC practice). | Inspect no-primary controls, blocking and wash steps, and detection exposure; rescore only after cell boundaries are interpretable (general IHC practice). |
| Only low or variable signal appears across otherwise usable sections. | HPA reports low tissue RNA specificity and low consistency between antibody staining and RNA expression (HPA: tissue IHC; HPA: reliability). The supplied sources do not identify an RBBP9-specific fixation sensitivity. | Compare the same named cell populations across sections and record intensity and proportion separately; use run controls to check assay performance (general IHC practice). |
| An IF/ICC result appears nucleoplasmic while IHC is cytoplasmic. | Those are the respective reported assay observations (HPA: subcellular ICC-IF; HPA: tissue IHC). | Record assay and compartment with each result; assess IF/ICC using its separate guide, without treating either pattern alone as proof of the other (HPA: subcellular ICC-IF; HPA: tissue IHC). |
Experiment evidence
IHC & IF images showing RBBP9 staining
A09378 · IHCImmunohistochemistry of paraffin-embedded rat ovary using RBBP9 Rabbit pAb at dilution of 1:200 .Perform high pressure antigen retrieval wit
A09378 · IHCImmunohistochemistry of paraffin-embedded rat kidney using RBBP9 Rabbit pAb at dilution of 1:200 .Perform high pressure antigen retrieval wi
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000089050-RBBP9/tissue/cerebral+cortex#imid_4277892
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000089050-RBBP9/tissue/colon#imid_4277826
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000089050-RBBP9/tissue/liver#imid_4277819
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000089050-RBBP9/tissue/kidney#imid_4277828
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA049005; green = RBBP9, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA049005; green = RBBP9, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RBBP9 IHC & IF
🧪Run adrenal gland first and expect staining in glandular cells (HPA: Medium in adrenal gland glandular cells). Use adipose tissue as the negative tissue, where adipocytes are listed as not detected (HPA: Not detected in adipocytes); on the adrenal slide, use morphologically unstained nonglandular areas to assess background without assuming those cells lack RBBP9.
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 RBBP9 in A-431, PC-3, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a host- and immunoglobulin-matched nonimmune rabbit IgG control (caption: Rabbit pAb); and a knockout specimen or validated peptide-block control. Quench endogenous peroxidase in adrenal sections for chromogenic detection and check the no-primary slide for pigment that could resemble staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A09378 paraffin-section caption does not state the fixative (caption: fixative not stated). That caption reports high-pressure retrieval in 10 mM citrate, pH 6.0, before staining rat ovary at 1:200; it does not establish that retrieval is required for adrenal gland (caption: rat ovary IHC). IF/ICC images exist for A-431, PC-3 and U2OS with supported nucleoplasmic localization, but the supplied evidence does not establish whether IF/ICC or frozen sections are easier (HPA: subcellular).
HPA tissue IHC evidence for RBBP9
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 | Glandular cells | 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 → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Basal cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RBBP9 IHC Tips
Use the catalog antibody’s paraffin-section image as a starting point, then judge staining against tissue morphology and the differing localisation reported by HPA.
How should I retrieve RBBP9 in paraffin sections when staining is weak?
Begin with heat-mediated retrieval in 10 mM citrate buffer at pH 6.0 (datasheet A09378). The catalog antibody’s rat ovary paraffin image used high-pressure retrieval with that buffer before staining at 1:200 (caption A09378). Keep section thickness, heating, and cooling consistent across comparison slides so retrieval differences do not masquerade as expression differences (standard IHC practice). If staining remains weak, test a different retrieval duration on adjacent sections before considering another buffer or pH as a fallback (standard IHC practice). Compare signal with tissue morphology and a no-primary control, since stronger colour alone does not establish specific RBBP9 staining (standard IHC practice).
Could fixation explain inconsistent RBBP9 staining between paraffin blocks?
Target-specific fixation sensitivity for RBBP9 is unknown from the supplied evidence, so treat fixation history as a variable to test rather than a documented cause. The rat ovary image identifies paraffin embedding but does not report its fixative or fixation time (caption A09378). For newly prepared material, record fixative, tissue thickness, and time in fixative; apply the same conditions to comparison samples (standard IHC practice). If archived blocks differ, stain adjacent sections together using citrate at pH 6.0 and the same detection run (datasheet A09378; standard IHC practice). Compare morphology and no-primary controls before attributing a signal difference to RBBP9 abundance (standard IHC practice).
Should RBBP9 staining be cytoplasmic or nuclear in tissue sections?
Expect cytoplasmic staining as a useful tissue-IHC reference because HPA reports cytoplasmic expression in most tissues (HPA tissue IHC). HPA separately reports supported nucleoplasmic localisation from ICC/IF images, so a nuclear component warrants evaluation rather than automatic rejection (HPA subcellular). UniProt supplies no subcellular annotation for RBBP9, leaving the compartment difference unresolved by that record (UniProt O75884). In glandular regions, compare staining with cell boundaries and nuclei on the counterstained section; HPA reports medium glandular-cell staining in several tissues (HPA tissue IHC; standard IHC practice). Score cytoplasmic and nuclear signal separately, and check each pattern against controls before interpreting either as specific (standard IHC practice).
Can the catalog antibody distinguish RBBP9 isoforms in paraffin sections?
UniProt lists 2 RBBP9 isoforms, but the supplied evidence does not map this antibody’s epitope to either isoform (UniProt O75884; supplied catalog evidence). Therefore, do not assign a stained cell to isoform 1 or 2 from chromogenic IHC alone (UniProt O75884; standard IHC interpretation). RBBP9 has no annotated transmembrane segment, signal peptide, glycosylation sites, or modified residues, which does not establish how fixation affects this antibody’s epitope (UniProt O75884). Compare the staining pattern with an independently validated reagent or an orthogonal expression measurement when isoform specificity matters (standard IHC practice). Report the antibody identity, retrieval conditions, and any remaining isoform ambiguity alongside the images (standard IHC reporting practice).
How should I assess RBBP9 by multiplex immunofluorescence?
Treat IF/ICC as a separate assay: HPA reports supported nucleoplasmic localisation in its cell images, while its tissue IHC profile is predominantly cytoplasmic (HPA subcellular; HPA tissue IHC). Multiplex with a marker for the cell population being evaluated, such as a glandular-cell marker where glandular staining is expected, and verify channel separation with single-stain controls (HPA tissue IHC; standard IF practice). Choose a far-red fluorophore when the specimen has strong shorter-wavelength autofluorescence, and include an unstained section to measure that background (standard IF practice). For an intracellular or nucleoplasmic epitope, test mild permeabilisation, such as 0.1% Triton X-100 for 5 minutes, against an unpermeabilised control (HPA subcellular; standard IF practice). Because the antibody epitope is unmapped, establish IF signal and compartment specificity with assay-specific controls (supplied catalog evidence; standard IF practice).
What should I change when the whole section develops brown staining?
First inspect a no-primary control and a reagent-only area to separate detection background from antibody-dependent staining (standard chromogenic IHC practice). Block endogenous peroxidase before DAB development, for example with 3% hydrogen peroxide for 10 minutes if compatible with the detection system (standard chromogenic IHC practice). Apply an appropriate protein block, wash thoroughly, and titrate the primary antibody around the catalog image’s 1:200 dilution on adjacent sections (caption A09378; standard IHC practice). Shorten DAB development if colour also accumulates outside intact cells, recording the same endpoint for every comparison slide (standard chromogenic IHC practice). Interpret residual diffuse staining cautiously because HPA describes broad cytoplasmic tissue expression for RBBP9 (HPA tissue IHC).
How can I compare RBBP9 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue compartment and exclude folds, necrosis, and section edges before scoring, using the same rules for every slide (standard IHC quantification practice). Record the percentage of positive cells and intensity by cell population, then calculate an H-score from 0 to 300 if ordinal intensity scoring is reproducible (standard IHC quantification practice). Normalise counts to the number of evaluable cells, or report positive-cell density per mm² when tissue area is the relevant denominator (standard IHC quantification practice). Score nuclear and cytoplasmic signal separately because HPA’s tissue IHC and ICC/IF localisation summaries differ (HPA tissue IHC; HPA subcellular). Hold retrieval, antibody dilution, DAB development, imaging, and thresholds constant across the comparison set (standard IHC quantification practice).
How do I distinguish credible RBBP9 staining from artefact?
Look for signal in intact cells with interpretable morphology; HPA’s tissue-IHC reference is predominantly cytoplasmic and includes medium staining in glandular cells (HPA tissue IHC). A nuclear pattern needs separate scrutiny because HPA supports nucleoplasmic localisation by ICC/IF, whereas its tissue-IHC summary describes cytoplasmic expression (HPA subcellular; HPA tissue IHC). Check whether colour is confined to section edges, necrotic areas, or damaged cells, and compare those regions with adjacent intact tissue (standard IHC interpretation). A positive no-primary control suggests detection background, including possible endogenous peroxidase activity in a DAB workflow (standard chromogenic IHC practice). Corroborate unexpected cell or compartment patterns with an independent antibody or orthogonal expression evidence before calling them RBBP9-positive (standard IHC interpretation).
Boster reagents
Best RBBP9 / Serine hydrolase RBBP9 IHC Antibodies
A09378 has real IHC images of paraffin-embedded rat ovary, rat kidney and human lung cancer (catalog image captions); listed reactivity covers human, mouse and rat (catalog: reactivity).
Real IHC data
Anti-Putative hydrolase RBBP9 RBBP9 Antibody
Cat # A09378
A09378 is listed for IHC and has images from paraffin-embedded rat ovary, rat kidney and human lung cancer (catalog: applications; image captions). It is listed as reactive with human, mouse and rat, with no IF application or IF image reported (catalog: reactivity; applications; IF images).
Which to pick: Choose A09378 for paraffin-section IHC: its captions specify 1:200 and high-pressure retrieval in 10 mM citrate buffer at pH 6.0; the fixative is unreported (catalog image captions). A09378 is a rabbit polyclonal with listed human, mouse and rat reactivity, though its IHC images show only human and rat samples (catalog: host; dilution_raw; reactivity; image captions). No SKU here is listed for IF/ICC, so this payload does not support an IF/ICC pick (catalog: applications; IF images).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O75884 (RBBP9_HUMAN, Serine hydrolase RBBP9).
- Human Protein Atlas. RBBP9 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. RBBP9 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. RBBP9 antibody validation summary (2 antibodies).
- Retinoblastoma-binding Protein 9 Suppresses Intestinal Inflammation and Inflammation-induced Tumorigenesis in Mice. Cellular and molecular gastroenterology and hepatology 2025 — PMC11786897.
- PubMed PMID:9697699 — UniProt-cited evidence.
- PubMed PMID:12296629 — UniProt-cited evidence.
- PubMed PMID:17974005 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RBBP9 (Serine hydrolase RBBP9). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, heat-mediated (datasheet A09378). Validated IHC antibody: A09378 at 1:50-1:200 (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 rat ovary tissue; fixative not specified (datasheet A09378); 2. Fixation: Image fixative and duration unreported (datasheet A09378); 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 6.0 (datasheet A09378); 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-RBBP9, 1:50-1:200 (datasheet A09378); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A09378); Caveat: Antibody staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unspecified (UniProt); Regulation: Higher expression in carcinoma tissues (UniProt). Evidence: UniProt O75884, HPA tissue IHC · Approved, HPA ICC-IF · nucleoplasm., Datasheet A09378, PMC11786897. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RBBP9/immunohistochemistry-ihc-guide.