RBBP6 / E3 ubiquitin-protein ligase RBBP6 · IHC design guide

Design Immunohistochemistry for RBBP6

Plan RBBP6 staining in paraffin sections around the low nuclear tissue pattern (HPA tissue IHC). Use the catalog antibody’s 2–5 μg/ml IHC range (datasheet A04382-2), and interpret intensity with the reported staining–RNA inconsistency in mind (HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for RBBP6 (IHC for RBBP6): expected localisation Low nuclear staining in several tissues (HPA tissue IHC), antibody A04382-2, validated IHC image, and IHC protocol steps — Printable **RBBP6** IHC protocol sheet — expected localisation Low nuclear staining in several tissues (HPA tissue IHC), antibody A04382-2, controls and protocol steps. Open the full RBBP6 IHC guide →

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RBBP6 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	Low nuclear staining in several tissues (HPA tissue IHC)
	Staining pattern	Low nuclear staining across several tissue cell types (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A04382-2)
	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	Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A04382-2)
	Caveat	Antibody staining has low consistency with RNA expression (HPA tissue IHC)
	Regulation	Overexpressed in esophageal cancer (UniProt)
	Isoform / epitope	4 isoforms; epitope coverage is unspecified (UniProt)

Section 1

Recommended RBBP6 IHC & IF Protocols

The catalog antibody protocol is accompanied by four published RBBP6 IHC protocols covering tumor specimens and tissue sections (PMC6828677; PMC3684577; PMC7068609; PMC6138973).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded rat testis tissue; fixative not specified (datasheet A04382-2)
Fixation	Image fixative and duration unreported (datasheet A04382-2); 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 A04382-2); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A04382-2)
Primary antibody	Rabbit anti-RBBP6, 2-5 μg/ml (datasheet A04382-2)
Primary incubation	Overnight at 4 °C (datasheet A04382-2)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A04382-2)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	RBBP6-positive staining in glandular cells of appendix (HPA tissue IHC: Medium). HPA tissue profile: Low nuclear expression in several tissues. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet A04382-2). Published protocols report EDTA pH 9.0 and citrate pH 6.0 (PMC7068609; PMC6138973).

Section 2

What Is the Expected RBBP6 Staining Pattern?

RBBP6 is primarily nuclear, with nucleolar localisation also reported; UniProt additionally lists mitotic chromosomes and the centrosome (UniProt Q7Z6E9 subcellular location). In paraffin-section IHC, expect nuclear staining in selected cell populations, including appendix and duodenal glandular cells and bronchial respiratory epithelial cells (HPA tissue IHC). HPA rates tissue IHC “Approved” but reports low consistency between antibody staining and RNA expression (HPA tissue IHC). RBBP6 has no transmembrane segment (UniProt Q7Z6E9 topology).

What am I looking at on my slide?
Nuclear staining in appendix or duodenal glandular cells, or bronchial respiratory epithelial cells (HPA tissue IHC).	This fits the reported medium staining in those cell types and RBBP6’s nuclear localisation (HPA tissue IHC; UniProt Q7Z6E9 subcellular location). Judge intensity against cells on the same section and the controls; “medium” is an HPA observation, not a required score for every specimen.
Predominantly cytoplasmic staining with little nuclear signal.	Treat this as a localisation mismatch requiring validation: HPA describes low nuclear tissue expression (HPA tissue IHC). UniProt also lists the centrosome and mitotic chromosomes, so an occasional nonnuclear structure alone does not establish an artefact (UniProt Q7Z6E9 subcellular location).
Strong staining in adipocytes, bone-marrow hematopoietic cells or pancreatic exocrine cells.	HPA reports these populations as not detected in its tissue IHC survey (HPA tissue IHC). Check for cross-reactivity or endogenous chromogenic activity before calling them RBBP6-positive (general IHC practice). A different result should be verified rather than dismissed solely from the HPA survey.
Diffuse colour across cells, stroma or empty areas without a readable nuclear pattern.	Background limits localisation and scoring (general IHC practice). Review the no-primary control, blocking and detection conditions; diffuse colour cannot be interpreted as the low nuclear expression described by HPA (HPA tissue IHC; general IHC practice).
No signal in an appendix or bronchus section expected to provide a positive population.	HPA reports medium staining in appendix glandular cells and bronchial respiratory epithelial cells (HPA tissue IHC). First confirm those cells are present and that controls worked; a blank section may reflect assay failure, while HPA’s low RNA–staining consistency limits certainty for any individual specimen (HPA tissue IHC; general IHC practice).

💡Expected RBBP6 appearanceA credible positive is readable nuclear colour in the appropriate glandular or respiratory epithelial cells, potentially around HPA’s reported medium level; dominant diffuse colour or strong staining in HPA-undetected populations warrants a control check (HPA tissue IHC; general IHC practice).

How each factor affects the staining
Tissue and cell selection	HPA reports medium staining in several specified populations, low staining in others, and no detection in some (HPA tissue IHC). Select and score by cell type, not by whole-section colour; bone marrow RNA enrichment does not establish hematopoietic-cell IHC positivity (HPA tissue IHC).
Subcellular interpretation	Nucleus and nucleolus are listed by UniProt; HPA tissue IHC summarises low nuclear expression (UniProt Q7Z6E9 subcellular location; HPA tissue IHC). The reported chromosome and centrosome associations call for care when interpreting unusual puncta (UniProt Q7Z6E9 subcellular location).
Antibody evidence	Two listed antibodies have “Approved” IHC status, while HPA still reports low consistency with RNA expression (HPA antibodies; HPA tissue IHC). Approval supports use of the observed pattern as a reference, not automatic confirmation of every stained cell.
IF/ICC Q&A: what pattern should an IF image show?	HPA reports enhanced nuclear-speckle localisation in ICC-IF (HPA subcellular; HPA antibodies). This is a separate imaging observation; do not require individually resolved speckles in chromogenic paraffin IHC (HPA subcellular; general IHC practice).
Isoforms and processing	UniProt lists four RBBP6 isoforms, no signal peptide or propeptide, and no transmembrane segment (UniProt Q7Z6E9 isoforms, processing and topology). The payload gives no antibody epitope, so it cannot establish which isoforms a given stain detects.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Expected positive cells are blank.	The reference population may be absent from the section, or an IHC step may have failed (HPA tissue IHC; general IHC practice).	Confirm the relevant cells by morphology, inspect a known-positive section, then check retrieval and detection using the antibody’s IHC instructions (general IHC practice). No RBBP6-specific retrieval requirement is supplied.
All tissue compartments are uniformly brown.	Nonspecific reagent binding or endogenous detection activity can obscure localisation (general IHC practice).	Compare with a no-primary control; review blocking, washes and endogenous-activity quenching appropriate to the chromogen (general IHC practice). Interpret only a resolved cell-associated pattern.
Colour is mainly cytoplasmic.	The result differs from HPA’s nuclear tissue profile, although UniProt lists additional locations (HPA tissue IHC; UniProt Q7Z6E9 subcellular location).	Check counterstained nuclei and controls, then confirm with an independently validated antibody if the cytoplasmic result persists (general IHC practice).
A population listed as undetected stains strongly.	Cross-reactivity or endogenous activity is possible; HPA’s undetected call is specific to its survey (HPA tissue IHC; general IHC practice).	Identify the stained cell type, compare no-primary and known-positive controls, and verify the result independently before assigning RBBP6 expression (general IHC practice).
Signal varies across replicate sections.	Section composition or inconsistent processing may change the apparent score (general IHC practice); HPA also reports low RNA–staining consistency (HPA tissue IHC).	Compare the same cell population across sections and review section handling, retrieval and detection records (general IHC practice). Do not infer a RBBP6-specific fixation effect from this variation.
IF shows speckles, but IHC looks broadly nuclear.	HPA’s speckle call comes from ICC-IF, while its tissue IHC summary is nuclear (HPA subcellular; HPA tissue IHC).	Assess each image against its own assay reference and controls; score paraffin IHC by interpretable nuclear staining in the specified cells (HPA tissue IHC; general IHC practice).

Source: UniProt Q7Z6E9 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing RBBP6 staining

IHC analysis of RBBP6 using anti-RBBP6 antibody (A04382-2). RBBP6 was detected in a paraffin-embedded section of rat testis tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-RBBP6 Antibody (A04382-2) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A04382-2 · IHCIHC analysis of RBBP6 using anti-RBBP6 antibody (A04382-2). RBBP6 was detected in a paraffin-embedded section of rat testis tissue. Heat med

IF analysis of RBBP6 using anti-RBBP6 antibody (A04382-2) and anti-Beta Tubulin antibody (M01857-3). RBBP6 was detected in an immunocytochemical section of Hela cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-RBBP6 Antibody (A04382-2) and mouse anti-Beta Tubulin antibody (M01857-3) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) and Cy3 Conjugated Goat Anti-Mouse IgG (BA1031) were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A04382-2 · IFIF analysis of RBBP6 using anti-RBBP6 antibody (A04382-2) and anti-Beta Tubulin antibody (M01857-3). RBBP6 was detected in an immunocytochem

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000122257-RBBP6/tissue/cerebral+cortex#imid_16037035

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000122257-RBBP6/tissue/colon#imid_16036953

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000122257-RBBP6/tissue/liver#imid_16036962

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000122257-RBBP6/tissue/kidney#imid_16036955

HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA041725; green = RBBP6, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for RBBP6 IHC & IF

🧪Run appendix first: its glandular cells should stain at the HPA-reported Medium level (HPA: appendix, glandular cells, Medium). Use adipose tissue as the negative comparator because adipocytes are Not detected (HPA: adipose tissue, adipocytes, Not detected); adjacent nonglandular cells on the appendix slide can serve as an internal background reference only if they remain unstained, since their RBBP6 status is unreported in the supplied HPA row.

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 RBBP6 in HEK293, MCF-7, U2OS, U2OS, siRNA 1 (10x), U2OS, siRNA 1 (40x), U2OS, scrambled (10x), U2OS, scrambled (40x), NIH 3T3, with annotated localisation: Nuclear speckles (enhanced) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control, a matched rabbit IgG isotype control, and an RBBP6 knockout control where available (caption: rabbit primary; standard IHC controls). Quench endogenous peroxidase before DAB detection so peroxidase background on the appendix slide is not scored as RBBP6 staining (caption: HRP/DAB detection; standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A04382-2 rat-testis paraffin-section caption does not state the fixative (selected-SKU caption: fixative not stated). That caption reports heat-mediated EDTA retrieval at pH 8.0, but does not establish that retrieval is required for every specimen (selected-SKU caption: EDTA retrieval, pH 8.0). The supplied evidence does not establish whether frozen sections or IF are easier; for appendix IHC, assess glandular staining against adjacent tissue background and the peroxidase control (HPA: appendix glandular cells, Medium; standard IHC practice).

HPA tissue IHC evidence for RBBP6

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
Appendix	Glandular cells	Medium	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	Medium	Protein (IHC)	HPA →
Cerebellum	Cells in granular layer	Medium	Protein (IHC)	HPA →
Cerebral cortex	Neuronal cells	Medium	Protein (IHC)	HPA →
Cervix	Squamous epithelial cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Adrenal gland	Glandular cells	Not detected	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →
Hippocampus	Glial cells	Not detected	Protein (IHC)	HPA →
Lymph node	Germinal center cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced RBBP6 IHC Tips

Troubleshoot RBBP6 staining in paraffin sections by checking retrieval, cellular localisation and controls before comparing chromogenic scores (datasheet A04382-2; UniProt Q7Z6E9).

Which retrieval condition should I try first for RBBP6 paraffin IHC?

Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for chromogenic paraffin IHC (datasheet A04382-2). The demonstrated section used a 2 μg/ml primary antibody overnight at 4°C, so hold those conditions steady while assessing retrieval (datasheet A04382-2). If nuclear staining is weak, adjust heating duration in small steps on adjacent sections, recording tissue damage and background alongside signal (standard IHC practice). Only if that fails, compare a different retrieval buffer on matched sections, with no-primary controls and identical DAB development; a brighter section alone does not establish specificity (standard IHC practice).

Could fixation explain weak or inconsistent RBBP6 staining?

RBBP6-specific fixation sensitivity is unknown: the paraffin-section caption does not state a fixative (datasheet A04382-2). Record the fixative, time to fixation, fixation duration and processing history for every section before comparing staining (standard IHC practice). Compare matched sections with the same EDTA pH 8.0 retrieval and 2 μg/ml primary incubation overnight at 4°C, changing one processing variable at a time (datasheet A04382-2; standard IHC practice). Inspect nuclear morphology and tissue edges while reviewing weak signal, because processing artefacts can confound chromogenic IHC comparisons (standard IHC practice). Neither a tissue-expression pattern nor RBBP6's intracellular localisation establishes a fixation effect (HPA: tissue IHC profile; UniProt Q7Z6E9 localisation).

Where should convincing RBBP6 staining appear within a section?

Prioritise interpretable nuclear staining: RBBP6 is reported in the nucleus and nucleolus, while HPA describes low nuclear expression across several tissues (UniProt Q7Z6E9 localisation; HPA: tissue IHC profile). Chromosomal and centrosomal localisation is also reported, including association with mitotic chromosomes, but these patterns require suitable cellular resolution to assess (UniProt Q7Z6E9 localisation). Score nuclear and cytoplasmic DAB separately rather than treating any brown deposit as equivalent RBBP6 signal (standard IHC practice). Use a nuclear counterstain and compare cellular boundaries on adjacent sections; isolated diffuse extracellular staining warrants a background check (standard IHC practice).

How could isoforms or epitope accessibility affect this stain?

RBBP6 has 4 listed isoforms, and its DWNN domain spans residues 4–76 of the supplied protein record (UniProt Q7Z6E9 isoforms and domains). The supplied antibody caption does not identify its immunogen or epitope, so staining cannot be assigned to a particular isoform or domain (datasheet A04382-2). Antigen retrieval can alter access to fixed epitopes; evaluate adjacent sections with matched detection and a consistent EDTA pH 8.0 starting condition (datasheet A04382-2; standard IHC practice). RBBP6 has reported acetylation and phosphorylation sites, but their effects on this antibody's staining are unknown without epitope information and targeted validation (UniProt Q7Z6E9 modified residues).

How should I plan IF to investigate an ambiguous IHC pattern?

Use IF as a separate localisation check: HPA reports enhanced RBBP6 signal in nuclear speckles in its subcellular images (HPA: subcellular profile). For multiplexing, pair RBBP6 with a validated marker of the cell population under study, such as a cytokeratin marker when evaluating bronchial respiratory epithelium, and include a nuclear counterstain (HPA: medium in bronchus respiratory epithelial cells; standard IF practice). Choose fluorophores after checking the tissue's autofluorescence in unstained controls, and separate emission channels to assess bleed-through (standard IF practice). Because RBBP6 is intracellular with no transmembrane segment, permeabilise sufficiently to reach nuclear epitopes, while optimising against loss of morphology; the antibody epitope is unspecified (UniProt Q7Z6E9 topology and localisation; datasheet A04382-2).

What controls help distinguish RBBP6 signal from DAB background?

Run no-primary and secondary-only controls through the same chromogenic workflow to reveal detection-system background (standard IHC practice). Include a peroxidase block before HRP detection and keep DAB development time matched across test and control sections (standard IHC practice). The demonstrated assay used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and an HRP-linked secondary for 30 minutes at 37°C (datasheet A04382-2). If staining is diffuse, titrate primary concentration and review section edges and tissue folds before interpreting compartment-specific signal (standard IHC practice). Compare suspected nuclear staining with the counterstain, since RBBP6 has reported nuclear localisation (UniProt Q7Z6E9 localisation).

How should I quantify RBBP6 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and nuclear scoring rule before reviewing study groups, since HPA describes low nuclear expression across several tissues (HPA: tissue IHC profile; standard IHC practice). Report the percentage of positive nuclei and an H-score based on intensity categories 0–3, or positive-cell density per mm² when area is the intended denominator (standard IHC practice). Normalise counts to evaluable cells or viable tissue area, and analyse nuclear and cytoplasmic staining separately (standard IHC practice). Keep retrieval, antibody exposure, DAB development, imaging and thresholds consistent, then record excluded folds, edges and necrotic areas (standard IHC practice).

When is an apparent RBBP6-positive cell credible?

A credible result shows reproducible cellular staining in the expected compartment, supported by clean detection controls and preserved morphology (UniProt Q7Z6E9 localisation; standard IHC practice). Give nuclear signal particular weight, while assessing cytoplasmic or mitotic patterns against the reported nucleolar, chromosome and centrosome localisations rather than assuming every brown structure is specific (UniProt Q7Z6E9 localisation). Check that the stained cell type is identified correctly; HPA reports medium staining in bronchial respiratory epithelial cells but also warns of low consistency between antibody staining and RNA data (HPA: tissue IHC profile and reliability). Edge-only signal, necrosis-associated deposits or staining present without primary antibody calls for renewed artefact and endogenous-enzyme checks before biological interpretation (standard IHC practice).

Boster reagents

Best RBBP6 / E3 ubiquitin-protein ligase RBBP6 IHC Antibodies

A04382-2 has IHC data from rat testis paraffin sections and IF/ICC data from HeLa cells; the catalog lists Human, Mouse and Rat reactivity (A04382-2 image captions; catalog reactivity).

Real IHC data

Anti-RBBP6 Antibody ®

Cat # A04382-2

View product →

A04382-2 was demonstrated by chromogenic IHC on a rat testis paraffin section (A04382-2 IHC image caption). The same SKU was demonstrated by IF/ICC in HeLa cells and is listed for IHC, ICC and IF with Human, Mouse and Rat reactivity (A04382-2 IF image caption; catalog applications and reactivity).

Which to pick: For tissue IHC, choose A04382-2: its rat testis paraffin-section image used EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody; the fixative is unreported (A04382-2 IHC image caption). For IF/ICC, A04382-2 has a HeLa-cell image using 5 μg/ml primary antibody (A04382-2 IF image caption). For cross-species work, A04382-2 lists Human, Mouse and Rat reactivity and a rabbit host; clonality is unspecified, and the supplied tissue IHC image demonstrates Rat only (A04382-2 catalog reactivity, host and clone fields; IHC image caption).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry Q7Z6E9 (RBBP6_HUMAN, E3 ubiquitin-protein ligase RBBP6).
Human Protein Atlas. RBBP6 tissue IHC expression (reliability: Approved).
Human Protein Atlas. RBBP6 subcellular location (ICC-IF): Localized to the nuclear speckles..
Human Protein Atlas. RBBP6 antibody validation summary (3 antibodies).
RBBP6, a RING finger-domain E3 ubiquitin ligase, induces epithelial-mesenchymal transition and promotes metastasis of colorectal cancer. Cell death & disease 2019 — PMC6828677.
Overexpression of RBBP6, alone or combined with mutant TP53, is predictive of poor prognosis in colon cancer. PloS one 2013 — PMC3684577.
RBBP6 induces non-small cell lung cancer cell proliferation and high expression is associated with poor prognosis. Oncology letters 2020 — PMC7068609.
RBBP6 expressional effects on cell proliferation and apoptosis in breast cancer cell lines with distinct p53 statuses. Cancer management and research 2018 — PMC6138973.
PubMed PMID:15489334 — UniProt-cited evidence.
PubMed PMID:8595913 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.