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RBBP8 / DNA endonuclease RBBP8 · IHC design guide
Design Immunohistochemistry for RBBP8
Plan chromogenic paraffin IHC around the nuclear and cytoplasmic staining reported in most tissues (HPA tissue IHC). Use medium-staining breast glandular cells and adipocytes with no detected staining as comparison populations (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RBBP8 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 | Nuclear and cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Nuclear and cytoplasmic staining across most tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A02076-2) | |
| Positive control | Appendix+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 | Splice/transcript discrepancies complicate staining–RNA comparison (HPA tissue IHC) | |
| Regulation | Tends lower in ER-negative breast lines (UniProt) | |
| Isoform / epitope | 3 isoforms; check epitope coverage for each (UniProt) |
Section 1
Recommended RBBP8 IHC & IF Protocols
The catalog antibody uses EDTA pH 8.0 heat retrieval (datasheet A02076-2). Two published RBBP8 IHC protocols provide low-pH heat and HCl/trypsin retrieval methods (PMC5802064; PMC3892382).
| Sample | Paraffin-embedded human endometrioid adenocarcinoma tissue; fixative not specified (datasheet A02076-2) |
| Fixation | Image fixative and duration unreported (datasheet A02076-2); 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 A02076-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A02076-2) |
| Primary antibody | Rabbit anti-RBBP8, 2-5 μg/ml (datasheet A02076-2) |
| Primary incubation | Overnight at 4 °C (datasheet A02076-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A02076-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RBBP8-positive staining in glandular cells of appendix (HPA tissue IHC: Medium). HPA tissue profile: Nuclear and cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A02076-2); evaluate the published retrieval methods with their respective antibodies (PMC5802064; PMC3892382).
Section 2
What Is the Expected RBBP8 Staining Pattern?
RBBP8 is a nuclear and chromosome-associated protein without a transmembrane segment (UniProt Q99708: subcellular location and topology). In paraffin-section IHC, expect nuclear staining, with possible cytoplasmic staining, in many tissues; HPA reports medium staining in several glandular epithelia and kidney tubular cells (HPA tissue IHC). The HPA IHC profile is Approved, with medium consistency against RNA data and a splice or transcript discrepancy, so interpret staining with controls (HPA tissue IHC).
| Glandular-cell nuclei stain in breast, colon, or appendix; some cytoplasmic color may accompany them (HPA tissue IHC). | This fits HPA's medium staining in those glandular cells and its broader nuclear and cytoplasmic tissue profile (HPA tissue IHC). Judge the result by the identified cells and compartments, rather than expecting identical color across every cell in the section (HPA tissue IHC: low tissue specificity; general IHC practice). |
| Color is confined to cell membranes or extracellular material, with no convincing nuclear staining. | A membrane-only or extracellular pattern does not fit RBBP8's nuclear and chromosome annotation or its lack of a transmembrane segment (UniProt Q99708). Check section morphology, detection background, and a known-positive control before assigning that color to RBBP8 (general IHC practice). Cytoplasmic color alone needs a more cautious judgment because HPA reports cytoplasmic tissue staining (HPA tissue IHC). |
| The strongest color appears in a cell population recorded as not detected, such as adipocytes or cardiomyocytes (HPA tissue IHC). | That distribution conflicts with the cited HPA observations for those cells, though HPA's Approved profile has medium RNA concordance and a splice or transcript discrepancy (HPA tissue IHC). Consider cross-reactivity or endogenous detection activity, then compare the suspect cells with an appropriate control section and a no-primary control (general IHC practice). |
| A uniform brown haze covers nuclei, cytoplasm, and tissue spaces, obscuring cell boundaries. | A haze without a readable cellular pattern cannot establish the reported RBBP8 distribution (HPA tissue IHC: nuclear and cytoplasmic profile; general IHC practice). Inspect the no-primary control and detection reagents; assess washing and blocking as general chromogenic IHC checks (general IHC practice). Avoid scoring diffuse color as positive cells until compartment boundaries are visible (general IHC practice). |
| No cellular signal is visible in glandular cells of a breast, colon, or appendix control section (HPA tissue IHC). | HPA records medium staining in those cell populations, so a blank control calls for review of the IHC workflow and antibody suitability (HPA tissue IHC; general IHC practice). Confirm the intended cells are present, inspect counterstain and detection controls, and compare sections processed in the same run (general IHC practice). A single blank field does not establish biological absence (general IHC practice). |
💡Expected RBBP8 appearanceCall a positive result when identifiable glandular or kidney tubular cells show interpretable nuclear staining, with possible cytoplasmic color and medium staining reported in the cited HPA tissues; membrane-only or structureless diffuse color is suspect (HPA tissue IHC; UniProt Q99708 topology; general IHC practice).
| IHC evidence and its limit | HPA rates tissue IHC Approved, with medium staining–RNA consistency and a splice or transcript discrepancy (HPA tissue IHC). Treat its cell-level observations as reference patterns, while keeping the discrepancy in mind when a sample differs (HPA tissue IHC). |
| Cell population and tissue | Medium staining is reported in glandular cells of appendix, breast, cervix, colon, duodenum, and gallbladder, plus esophageal squamous cells and kidney tubular cells (HPA tissue IHC). HPA records no detection in adipocytes, cardiomyocytes, and skeletal myocytes, among other listed cells (HPA tissue IHC). |
| Localization and damage context | UniProt places RBBP8 in the nucleus and on chromosomes and describes recruitment to DNA-damage sites in S/G2 (UniProt Q99708). HPA tissue IHC also reports cytoplasmic staining (HPA tissue IHC). Do not infer DNA damage, cell-cycle phase, or specificity from an ordinary chromogenic pattern alone (general IHC interpretation). |
| Isoforms and antibody evidence | UniProt lists three RBBP8 isoforms (UniProt Q99708). The payload supplies no epitope map or isoform-specific IHC result, so this section cannot assign a staining difference to one isoform. HPA039890 has Approved IHC status; HPA052946 has Supported ICC status, which is a separate application (HPA antibodies). |
| IF/ICC Q: Where should fluorescence appear? | A: Mainly in the nucleoplasm, based on HPA's supported subcellular location; HPA lists ICC-IF images for MCF-7, U-251MG, and U2OS (HPA subcellular). That ICC-IF observation helps interpret localization but does not establish a paraffin-section IHC protocol or guarantee identical appearance in tissue (HPA subcellular; general IHC/IF practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive tissue is blank or unusually faint. | The observed result differs from HPA's medium glandular-cell or kidney tubular-cell staining; the cause cannot be assigned from that difference alone (HPA tissue IHC). | Verify that the cited cell population is present and review the IHC-validated antibody, antigen retrieval, primary incubation, and detection controls as general paraffin IHC checks (HPA antibodies; general IHC practice). No RBBP8-specific retrieval condition is supplied. |
| Signal is predominantly at membranes or in tissue spaces. | That location conflicts with UniProt's nuclear and chromosome annotation and lack of a transmembrane segment (UniProt Q99708). | Inspect a no-primary control and tissue morphology, then reassess detection background before scoring (general IHC practice). Retain a mixed nuclear and cytoplasmic result as a possibility because HPA reports both compartments in tissue (HPA tissue IHC). |
| Cells listed as not detected appear strongly positive. | The result differs from HPA's observations for adipocytes, cardiomyocytes, or skeletal myocytes; cross-reactivity or endogenous detection activity is possible (HPA tissue IHC; general IHC practice). | Check cell identity and compare a no-primary control with known-positive cells in the same run (general IHC practice). Record the mismatch rather than treating a single HPA entry as proof of biological absence (HPA tissue IHC: Approved, medium consistency). |
| Diffuse brown background obscures nuclei. | Nonspecific detection or incomplete washing can obscure compartment-level interpretation (general chromogenic IHC practice). | Inspect no-primary and detection controls, then review blocking, wash steps, and chromogen development using the assay's established workflow (general IHC practice). Score only cells whose boundaries and nuclear signal remain interpretable (general IHC practice). |
| Sections show patchy staining across otherwise comparable fields. | HPA reports low tissue specificity but varied cell-level staining, including medium, low, and not-detected entries; a patchy field alone has no single established cause (HPA tissue IHC). | Compare like cell populations across fields, confirm section quality, and check that control sections were processed together (general IHC practice). Do not attribute the variation to RBBP8 fixation sensitivity; no target-specific fixation evidence is supplied. |
| IHC and ICC-IF images seem to disagree about cytoplasmic signal. | HPA describes nuclear and cytoplasmic tissue IHC but mainly nucleoplasmic ICC-IF; the observations come from different applications (HPA tissue IHC; HPA subcellular). | Interpret each result against its own HPA application evidence and controls (HPA tissue IHC; HPA subcellular; general IHC/IF practice). For the paraffin-section decision, identify stained tissue cells and require an interpretable nuclear component. |
Experiment evidence
IHC & IF images showing RBBP8 staining
A02076-2 · IHCIHC analysis of RBBP8 using anti-RBBP8 antibody (A02076-2). RBBP8 was detected in a paraffin-embedded section of human endometrioid adenocar
A02076-2 · IHCIHC analysis of RBBP8 using anti-RBBP8 antibody (A02076-2). RBBP8 was detected in a paraffin-embedded section of human mucinous adenoma of o
A02076-2 · IFIF analysis of RBBP8 using anti-RBBP8 antibody (A02076-2) and anti-Beta Tubulin antibody (M01857-3). RBBP8 was detected in immunocytochemica
A02076 · IHCImmunohistochemistry analysis of paraffin-embedded human lung carcinoma tissue, using CTIP Antibody. The picture on the right is blocked wit
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000101773-RBBP8/tissue/cerebral+cortex#imid_10919754
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000101773-RBBP8/tissue/colon#imid_10824153
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000101773-RBBP8/tissue/liver#imid_10824146
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000101773-RBBP8/tissue/kidney#imid_10824147
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA052946; green = RBBP8, blue = nucleus, red = microtubules.
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA052946; green = RBBP8, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RBBP8 IHC & IF
🧪Run breast first and look for staining in glandular cells (HPA: Medium in breast glandular cells). Use adipose tissue as a negative comparator (HPA: Not detected in adipocytes); on the breast slide, assess any adjacent adipocytes separately and expect little signal relative to glandular cells, while confirming background with the no-primary control (HPA: Not detected in adipocytes in adipose tissue).
Positive control tissue: Appendix (Glandular cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RBBP8 in MCF-7, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host-species-matched isotype controls, plus RBBP8 knockout material or a validated peptide-block control to assess specificity (standard IHC practice). For chromogenic detection, quench endogenous peroxidase and inspect the breast section for residual background before scoring (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected A02076-2 tissue-IHC caption does not state a fixative (selected-SKU caption). Heat retrieval in EDTA at pH 8.0 is a documented starting condition for paraffin sections (selected-SKU caption); the evidence does not establish that frozen sections or IF are easier. For IF, check breast tissue autofluorescence with an unstained section before interpreting nuclear signal (standard IF practice); nucleoplasmic localization is the expected pattern (HPA: Nucleoplasm, supported).
HPA tissue IHC evidence for RBBP8
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Caution, Splice and/or transcript discrepancy exists.). 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Colon | Glandular cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RBBP8 IHC Tips
Troubleshoot RBBP8 staining in paraffin sections by checking retrieval, compartment, controls and scoring before interpreting differences between samples.
How should I optimize retrieval when nuclear RBBP8 staining is weak?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A02076-2). The selected tissue-IHC example used this retrieval before 2 μg/ml primary antibody overnight at 4°C, providing a matched starting condition (datasheet A02076-2). If staining is weak, compare retrieval heating times such as 10 and 20 minutes on adjacent sections while holding antibody concentration and detection constant (standard IHC practice). Judge recovery by nuclear signal relative to background, since RBBP8 is mainly nucleoplasmic and also associates with chromosomes at DNA-damage sites (HPA subcellular; UniProt Q99708). Avoid interpreting stronger overall brown staining alone as improved retrieval (standard IHC practice).
Could fixation explain loss of RBBP8 signal in my paraffin sections?
Target-specific fixation sensitivity is unknown because the selected paraffin-section caption does not report a fixative or fixation duration (datasheet A02076-2). Record the fixative and time for each specimen, then compare similarly processed sections before attributing a weak result to biological variation (standard IHC practice). Keep retrieval at EDTA pH 8.0 and the primary antibody at 2 μg/ml while assessing that comparison (datasheet A02076-2). Include a positive tissue control processed alongside the samples, and check whether its expected nuclear signal also falls (HPA subcellular; standard IHC practice). Tissue staining patterns cannot establish which fixation condition preserves this antibody’s epitope (standard IHC practice).
Should RBBP8 staining appear only in nuclei?
Prioritize nucleoplasmic staining when assessing a paraffin-section result, because supported subcellular data place RBBP8 mainly in the nucleoplasm (HPA subcellular). Nuclear or chromosomal enrichment is also consistent with its association with DNA-damage sites during S/G2 (UniProt Q99708). Tissue-IHC data additionally describe nuclear and cytoplasmic staining in most tissues, so cytoplasmic color alone is insufficient to call a section negative or positive (HPA tissue IHC). Compare each compartment with adjacent cells, a negative detection control and the same exposure to chromogen (standard IHC practice). If signal is exclusively diffuse cytoplasmic, recheck blocking, antibody concentration and nuclear counterstain before assigning RBBP8 localisation (HPA subcellular; standard IHC practice).
Can this antibody distinguish RBBP8 isoforms or phosphorylation states in tissue?
Do not assign an isoform from chromogenic staining alone: RBBP8 has 3 annotated isoforms, while the supplied IHC caption does not identify this antibody’s epitope (UniProt Q99708; datasheet A02076-2). The canonical chain spans residues 1–897, and annotated modifications include phosphorylation at Thr-315 and Ser-327 (UniProt Q99708). Those sites do not establish whether fixation or retrieval changes this antibody’s binding, because target-specific epitope and fixation evidence is absent (UniProt Q99708; datasheet A02076-2). For an isoform or phospho-state claim, first obtain epitope mapping and appropriate validated controls, then assess staining under the same EDTA pH 8.0 retrieval condition (datasheet A02076-2; standard IHC practice).
How can I check RBBP8 localisation by multiplex IF?
Use IF as an independent localisation check alongside the chromogenic IHC section, selecting a marker for the expected cell population in the specimen (standard IF practice). Pair that marker with a nuclear counterstain and assess whether RBBP8 signal occupies nucleoplasm, its supported main location (HPA subcellular; standard IF practice). Choose fluorophores in channels with low measured tissue autofluorescence, and include unstained and single-label controls to evaluate bleed-through (standard IF practice). RBBP8 has no transmembrane segment, so optimize permeabilisation for access to an intracellular, predominantly nuclear epitope rather than assuming a cell-surface staining condition will work (UniProt Q99708; HPA subcellular; standard IF practice). Validate IF fixation and antibody performance separately; the paraffin IHC caption supplies no IF fixation condition (datasheet A02076-2).
What should I change when DAB background obscures nuclear staining?
Compare the specimen with a no-primary control to separate antibody-associated signal from detection background (standard IHC practice). The selected paraffin-section example used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and peroxidase detection with DAB (datasheet A02076-2). If background persists, verify an effective endogenous peroxidase block, adjust primary concentration and washing, and shorten chromogen development one variable at a time (standard IHC practice). Examine tissue edges and damaged areas separately because concentrated chromogen there can mimic specific staining (standard IHC practice). Require interpretable nuclear signal above the no-primary control before scoring RBBP8, whose supported main location is nucleoplasmic (HPA subcellular; standard IHC practice).
How should I score RBBP8 across heterogeneous tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and nuclear scoring rule before reviewing experimental groups, because RBBP8 is mainly nucleoplasmic but tissue IHC also reports cytoplasmic staining (HPA subcellular; HPA tissue IHC; standard IHC practice). For nuclear DAB, record the percentage of positive eligible cells and intensity categories 0–3, then calculate an H-score on the 0–300 scale (standard IHC practice). Normalize counts to the number of intact cells in the predefined population, or report positive-cell density per mm² of evaluable tissue (standard IHC practice). Exclude necrosis, folds and section edges by the same rule in every specimen (standard IHC practice). Report cytoplasmic staining separately so it does not inflate the nuclear score (HPA subcellular; standard IHC practice).
How do I distinguish true RBBP8 staining from artefact?
Give greatest weight to reproducible nucleoplasmic staining in intact cells, consistent with the supported main location of RBBP8 (HPA subcellular; standard IHC practice). Match the stained cells to tissue context: glandular cells in breast and colon have medium tissue-IHC staining, whereas adipocytes are reported as not detected (HPA tissue IHC). Diffuse color confined to section edges, necrotic regions or a no-primary control warrants investigation before calling a sample positive (standard IHC practice). Check endogenous peroxidase activity when DAB appears without convincing cellular localisation, and compare the finding with a processed positive control (standard IHC practice). Treat disagreements cautiously because the tissue-IHC dataset has medium RNA–staining consistency and a splice or transcript discrepancy warning (HPA tissue IHC).
Boster reagents
Best RBBP8 / DNA endonuclease RBBP8 IHC Antibodies
Catalog images show anti-RBBP8 IHC in human paraffin tissue sections and IF/ICC in HeLa cells; both antibodies list human and mouse reactivity (catalog image captions and reactivity lists).
Real IHC data
Anti-RBBP8 Antibody ®
Cat # A02076-2
Real IHC data
Anti-CTIP RBBP8 Antibody
Cat # A02076
A02076-2 has IHC images of human endometrioid adenocarcinoma, ovarian mucinous adenoma, and spleen paraffin sections, plus an IF/ICC image of HeLa cells (A02076-2 image captions). A02076 has an IHC image of a human lung carcinoma paraffin section with peptide blocking shown; IF/ICC is listed as an application without an IF image (A02076 image caption and application list).
Which to pick: For tissue IHC, choose A02076-2 when you want a documented starting condition: its paraffin-section caption specifies EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody; A02076 offers a paraffin-section image with peptide blocking and a listed IHC dilution of 1:100–1:300 (A02076-2 and A02076 IHC captions; A02076 datasheet). For IF/ICC, A02076-2 has a HeLa image at 5 μg/ml, while A02076 lists IF/ICC without an IF image (A02076-2 IF caption; A02076 application list). Both list human and mouse reactivity, though the supplied images show human samples only; neither IHC caption reports the fixative (catalog reactivity lists and image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q99708 (CTIP_HUMAN, DNA endonuclease RBBP8).
- Human Protein Atlas. RBBP8 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. RBBP8 subcellular location (ICC-IF): Mainly localized to the nucleoplasm..
- Human Protein Atlas. RBBP8 antibody validation summary (2 antibodies).
- Promoter methylation of DNA damage repair (DDR) genes in human tumor entities: RBBP8/CtIP is almost exclusively methylated in bladder cancer. Clinical epigenetics 2018 — PMC5802064.
- Prognostic value of CtIP/RBBP8 expression in breast cancer. Cancer medicine 2013 — PMC3892382.
- The mechanism of radiotherapy for lung adenocarcinoma in promoting protein SIRT6-mediated deacetylation of RBBP8 to enhance the sensitivity of targeted therapy. International journal of immunopathology and pharmacology 2022 — PMC9523831.
- Neoadjuvant palbociclib on ER+ breast cancer (N007): clinical response and EndoPredict's value. Endocrine-related cancer 2018 — PMC5763422.
- PubMed PMID:9721205 — UniProt-cited evidence.
- PubMed PMID:9535825 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RBBP8 (DNA endonuclease RBBP8). Expected localisation Nuclear and cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A02076-2). Validated IHC antibody: A02076-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human endometrioid adenocarcinoma tissue; fixative not specified (datasheet A02076-2); 2. Fixation: Image fixative and duration unreported (datasheet A02076-2); 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 A02076-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A02076-2); 8. Primary antibody: Rabbit anti-RBBP8, 2-5 μg/ml (datasheet A02076-2); 9. Primary incubation: Overnight at 4 °C (datasheet A02076-2). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Splice/transcript discrepancies complicate staining–RNA comparison (HPA tissue IHC); Isoform / epitope: 3 isoforms; check epitope coverage for each (UniProt); Regulation: Tends lower in ER-negative breast lines (UniProt). Evidence: UniProt Q99708, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm., Datasheet A02076-2, PMC5802064, PMC3892382. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RBBP8/immunohistochemistry-ihc-guide.