RNF123 / E3 ubiquitin-protein ligase RNF123 · IHC design guide

Design Immunohistochemistry for RNF123

Plan RNF123 IHC in paraffin sections around the general cytoplasmic tissue pattern (HPA tissue IHC). Compare high-staining kidney tubules or colon glandular cells with adipocytes reported as undetected, and interpret differences with the medium consistency between staining and RNA 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 RNF123 (IHC for RNF123): expected localisation Cytoplasmic staining (HPA tissue IHC)., antibody A09642-1, validated IHC image, and IHC protocol steps — Printable **RNF123** IHC protocol sheet — expected localisation Cytoplasmic staining (HPA tissue IHC)., antibody A09642-1, controls and protocol steps. Open the full RNF123 IHC guide →

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RNF123 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 (HPA tissue IHC).
	Staining pattern	General cytoplasmic staining across tissue cell types (HPA tissue IHC).
	Antigen retrieval	Citrate pH 6 HIER, heat-mediated (datasheet A09642-1)
	Positive control ⓘ	Cerebellum+4 more · see all
	Negative control ⓘ	Adipose tissue+3 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	Staining and RNA levels show medium consistency (HPA tissue IHC).
	Regulation	Expression regulation is not annotated (UniProt).
	Isoform / epitope	Two isoforms; epitope differences are unspecified (UniProt).

Section 1

Recommended RNF123 IHC & IF Protocols

The catalog antibody’s IHC-P protocol is paired with one published RNF123 IHC protocol using human FFPE tissue microarrays (PMC7281601).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human intestinal cancer tissues; fixative not specified (datasheet A09642-1)
Fixation	Image fixative and duration unreported (datasheet A09642-1); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: Citrate pH 6, 20 min (datasheet A09642-1)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A09642-1)
Primary antibody	Rabbit anti-RNF123, 0.5-1μg/ml (datasheet A09642-1)
Primary incubation	Overnight at 4 °C (datasheet A09642-1)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A09642-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	RNF123-positive staining in cells in molecular layer of cerebellum (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control.

💡Decision noteStart with heat-mediated citrate pH 6 retrieval for the catalog antibody (datasheet A09642-1); the published protocol does not specify retrieval (PMC7281601).

Section 2

What Is the Expected RNF123 Staining Pattern?

RNF123 should appear mainly in the cytoplasm, with a cytosolic pattern supported by ICC-IF (UniProt Q5XPI4: cytoplasm; HPA: approved cytosol). In paraffin-section IHC, high staining is reported in colon and rectal glandular cells, kidney tubular cells, and testis Leydig cells (HPA: High). HPA describes general cytoplasmic expression and rates its tissue IHC reliability Enhanced, while noting medium consistency between antibody staining and RNA expression (HPA: tissue IHC).

What am I looking at on my slide?
Cytoplasmic staining in colon or rectal glandular cells, or kidney tubular cells (HPA: High).	This matches the reported compartment and cell patterns (HPA: general cytoplasmic expression; HPA: High in these cells). Score the relevant cells against nearby unstained areas and the section's background; do not treat staining throughout the section as equally informative (general IHC practice).
Predominantly nuclear staining, with little convincing cytoplasmic signal (HPA: approved cytosol).	That distribution conflicts with the expected RNF123 location (UniProt Q5XPI4: cytoplasm; HPA: approved cytosol). Check whether counterstain, precipitated chromogen, or nonspecific detection is being mistaken for target staining before assigning a positive score (general IHC practice).
Strong staining in adipocytes, ovarian stromal cells, or chondrocytes (HPA: Not detected).	These cells are poor positive references because HPA reports no detected staining in them (HPA: tissue IHC). Unexpected signal warrants a specificity check; cross-reactivity or endogenous detection activity are possible explanations, not conclusions established by HPA (general IHC practice).
Similar diffuse color over cells, stroma, and empty areas of the section.	A diffuse field is difficult to attribute to cytoplasmic RNF123 (HPA: general cytoplasmic expression). Review blocking, washing, detection reagent, and chromogen development, then compare a matched control processed without primary antibody (general IHC practice).
No convincing cytoplasmic signal in colon glandular cells or kidney tubular cells (HPA: High).	This is discordant with the reported high staining, although a single section cannot establish target absence (HPA: tissue IHC). Inspect tissue preservation and assay controls, then review the IHC-validated antibody's stated conditions before interpreting other negative samples (general IHC practice).

💡Expected RNF123 appearanceCall an IHC result positive when distinct cytoplasmic staining is evident in reported high-staining glandular or tubular cells (HPA: High; HPA: general cytoplasmic expression); predominantly nuclear color or uniform diffuse background is suspect (HPA: approved cytosol; general IHC practice).

How each factor affects the staining
Compartment and topology (UniProt Q5XPI4: cytoplasm; no transmembrane segment).	RNF123 is expected within cells rather than as a membrane outline (UniProt Q5XPI4 topology; HPA: approved cytosol). Interpret chromogenic color in the context of cell boundaries and counterstain (general IHC practice).
Cell-specific reference patterns (HPA: tissue IHC).	High staining is reported in colon and rectal glandular cells, kidney tubular cells, and testis Leydig cells; adipocytes are Not detected (HPA: tissue IHC). Compare the named cell populations rather than treating a whole tissue as uniformly positive or negative (general IHC practice).
Validation and scope (HPA: tissue IHC; HPA: antibodies).	HPA labels tissue IHC reliability Enhanced but reports medium staining–RNA consistency; HPA065983 and HPA071879 each have Enhanced IHC status (HPA: tissue IHC; HPA: antibodies). These summaries support a reference pattern without guaranteeing that every positive-looking cell is specific.
IF/ICC interpretation (HPA: subcellular ICC-IF).	What should IF/ICC show? Mainly cytosolic RNF123; HPA lists PC-3 and SH-SY5Y images and an approved cytosol location (HPA: subcellular ICC-IF). This localisation reference does not supply an IF/ICC protocol or establish an IHC dilution.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A reported high-staining reference has no signal (HPA: High in colon glandular or kidney tubular cells).	The stain may have failed, or the selected section may lack well-preserved reference cells (general IHC practice). HPA's tissue rating does not identify the cause in an individual run (HPA: tissue IHC).	Confirm the named cells are present; review controls, retrieval, dilution, and detection against the IHC-validated antibody's documented conditions, then repeat as needed (general IHC practice).
Nuclear color dominates the slide (HPA: approved cytosol).	The distribution disagrees with the cytoplasmic reference pattern; counterstain or nonspecific chromogen may complicate reading (UniProt Q5XPI4: cytoplasm; general IHC practice).	Compare primary-omission and positive controls, inspect color localisation at higher magnification, and score cytoplasmic signal separately from nuclear counterstain (general IHC practice).
Adipocytes or ovarian stromal cells stain strongly (HPA: Not detected).	Unexpected staining could reflect cross-reactivity or endogenous detection activity; the HPA negative observations alone cannot distinguish them (HPA: tissue IHC; general IHC practice).	Check a matched control without primary antibody and the detection system's blocking steps; compare with a reported high-staining cell population in the same run (general IHC practice; HPA: tissue IHC).
Diffuse background obscures cell boundaries.	Excess detection signal, inadequate blocking or washing, or chromogen deposit can obscure the expected cytoplasmic pattern (general IHC practice; HPA: general cytoplasmic expression).	Review reagent controls and processing consistency; adjust blocking, washes, or development according to the validated assay instructions before rescoring (general IHC practice).
Only a faint signal appears in a low-staining reference cell population (HPA: Low in oral squamous cells).	Low staining is within HPA's reported range for oral mucosal squamous cells and does not by itself show assay failure (HPA: tissue IHC).	Judge performance using a reported high-staining population and controls; record intensity by cell type rather than calling the entire section negative (HPA: tissue IHC; general IHC practice).
IF/ICC appears concentrated outside the cytosol (HPA: approved cytosol).	The image conflicts with HPA's mainly cytosolic reference; optical overlap or nonspecific fluorescence may affect interpretation (HPA: subcellular ICC-IF; general IF practice).	Inspect separate fluorescence channels and controls, then compare the cellular distribution with HPA's cytosolic reference; use the dedicated IF/ICC guide for protocol choices (general IF practice; HPA: subcellular ICC-IF).

Source: UniProt Q5XPI4 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing RNF123 staining

IHC analysis of RNF123 using anti-RNF123 antibody (A09642-1). RNF123 was detected in paraffin-embedded section of human intestinal cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-RNF123 Antibody (A09642-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

A09642-1 · IHCIHC analysis of RNF123 using anti-RNF123 antibody (A09642-1). RNF123 was detected in paraffin-embedded section of human intestinal cancer ti

IHC analysis of RNF123 using anti-RNF123 antibody (A09642-1). RNF123 was detected in paraffin-embedded section of human lung cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-RNF123 Antibody (A09642-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

A09642-1 · IHCIHC analysis of RNF123 using anti-RNF123 antibody (A09642-1). RNF123 was detected in paraffin-embedded section of human lung cancer tissues.

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000164068-RNF123/tissue/cerebral+cortex#imid_18824262

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000164068-RNF123/tissue/colon#imid_18824180

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000164068-RNF123/tissue/liver#imid_18824181

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000164068-RNF123/tissue/kidney#imid_18824190

HPA · ICC-IF PC-3Human Protein Atlas immunofluorescence, PC-3, antibody HPA071879; green = RNF123, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for RNF123 IHC & IF

🧪Run colon first and confirm staining in glandular cells (HPA: High in colon glandular cells). Use adipose tissue as a negative comparator, focusing on adipocytes (HPA: Not detected in adipocytes); on the colon slide, use adjacent nonglandular cells as background comparators only if they remain unstained, since the HPA row does not establish that they are RNF123-negative.

Positive control tissue: Cerebellum (Cells in molecular layer, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RNF123 in PC-3, SH-SY5Y, with annotated localisation: Cytosol (approved) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control and a concentration-matched rabbit IgG isotype control (selected-SKU caption: rabbit primary antibody). Use RNF123 knockout tissue or a validated peptide block as a biological specificity control; quench endogenous peroxidase and assess endogenous biotin before using the caption’s streptavidin–biotin/DAB detection (selected-SKU caption: SABC with DAB).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative not stated). The caption uses citrate retrieval at pH 6 for 20 minutes, but does not establish that this retrieval is required (selected-SKU caption: citrate retrieval, pH 6, 20 minutes). There is no supplied basis to rank frozen sections or IF as easier; ICC-IF images show mainly cytosolic RNF123 in PC-3 and SH-SY5Y cells (HPA subcellular: cytosol, approved), while endogenous biotin may complicate the caption’s streptavidin–biotin detection in colon tissue.

HPA tissue IHC evidence for RNF123

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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
Cerebellum	Cells in molecular layer	High	Protein (IHC)	HPA →
Colon	Glandular cells	High	Protein (IHC)	HPA →
Epididymis	Glandular cells	High	Protein (IHC)	HPA →
Kidney	Cells in tubules	High	Protein (IHC)	HPA →
Rectum	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 →
Caudate	Glial cells	Not detected	Protein (IHC)	HPA →
Ovary	Ovarian stroma cells	Not detected	Protein (IHC)	HPA →
Soft tissue	Chondrocytes	Not detected	Protein (IHC)	HPA →

Section 3

Advanced RNF123 IHC Tips

Troubleshoot RNF123 staining in paraffin sections by checking retrieval, cytoplasmic localisation, cell identity and controls before comparing signal across samples.

What retrieval conditions should I start with for RNF123 in paraffin sections?

Start with heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet A09642-1). The selected image used paraffin-embedded human intestinal cancer tissue, followed by 10% goat-serum block and 1 μg/ml primary antibody overnight at 4°C (datasheet A09642-1). If staining is weak, compare a longer heat exposure or another retrieval buffer on adjacent sections while holding detection conditions constant (standard IHC practice). Include a colon glandular-cell section as a positive comparator and inspect tissue integrity, because stronger retrieval can increase background or damage morphology (HPA: High in colon glandular cells; standard IHC practice).

Could fixation explain weak or uneven RNF123 staining?

The selected paraffin-section caption does not state a fixative, so RNF123-specific sensitivity to fixation is unknown (datasheet A09642-1). Record fixative, fixation duration, processing history and section age for each specimen before attributing weak staining to the target (standard IHC practice). Compare sections processed together using the same citrate pH 6 retrieval for 20 minutes and identical antibody incubation (datasheet A09642-1; standard IHC practice). A colon glandular-cell control can reveal a run-wide loss of detectable signal, but it cannot establish which fixation condition preserves RNF123 best (HPA: High in colon glandular cells; standard IHC practice).

Which staining pattern is plausible for RNF123, and how should nuclear signal be assessed?

Expect predominantly cytoplasmic staining: RNF123 is annotated in the cytoplasm, and the subcellular profile places it mainly in the cytosol (UniProt Q5XPI4: subcellular location; HPA subcellular: Cytosol). It has no annotated transmembrane segment, so a crisp membrane-only pattern needs additional validation (UniProt Q5XPI4: topology). Compare stained cells with adjacent haematoxylin-stained nuclei and evaluate signal within cell boundaries rather than calling diffuse DAB deposits intracellular (standard IHC practice). If nuclear staining dominates, repeat the stain with a positive tissue control and appropriate detection controls before interpreting a change in RNF123 localisation (HPA: High in colon glandular cells; standard IHC practice).

Can this IHC stain distinguish RNF123 isoforms or modification states?

RNF123 has 2 annotated isoforms, but the supplied IHC caption does not identify the antibody epitope or establish isoform selectivity (UniProt Q5XPI4: isoforms; datasheet A09642-1). The protein contains a B30.2/SPRY region at residues 74–254 and annotated modifications at residues 2, 675 and 683 (UniProt Q5XPI4: domains and modified residues). Those annotations alone cannot show whether processing or modification changes antibody binding in a section (standard IHC interpretation). Check epitope documentation and validate any isoform-specific or modification-specific claim with a suitable independent assay before assigning a DAB pattern to one molecular form (standard IHC practice).

How should I adapt RNF123 localisation checks to multiplex IF?

For IF/ICC, seek cytosolic RNF123 signal and choose permeabilisation that gives antibody access to that intracellular compartment (HPA subcellular: Cytosol; standard IF practice). Pair RNF123 with a marker defining the expected cell population, such as a validated glandular-cell marker when examining colon, and include a nuclear counterstain for segmentation (HPA: High in colon glandular cells; standard IF practice). Select spectrally separated fluorophores and inspect unstained tissue for autofluorescence before assigning weak signal to RNF123 (standard IF practice). The supplied paraffin-section caption documents chromogenic detection, so optimise IF fixation, permeabilisation and antibody conditions independently rather than treating those IHC conditions as an IF protocol (datasheet A09642-1; standard IF practice).

What should I adjust when RNF123 DAB staining is diffuse or widespread?

First compare the pattern with the expected general cytoplasmic expression and examine whether DAB lies within intact cells (HPA tissue IHC: general cytoplasmic expression; standard IHC practice). The selected image used 10% goat serum, 1 μg/ml primary antibody overnight at 4°C, and biotin-based DAB detection (datasheet A09642-1). Use no-primary and secondary-only controls, block endogenous peroxidase, and assess endogenous biotin if retaining biotin-based detection (standard IHC practice). If controls are clean but staining remains diffuse, titrate primary antibody and review washing, retrieval exposure and section drying while keeping a positive control in the run (standard IHC practice).

How should I quantify RNF123 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and analysis region before scoring, then exclude folds, necrosis and tissue edges using the same criteria for every section (standard IHC practice). Score cytoplasmic intensity with an H-score, report the percentage of positive cells, or count positive cells per mm² when cell density matters (HPA tissue IHC: general cytoplasmic expression; standard IHC practice). Normalise positive-cell counts to the number of evaluable cells or sampled tissue area, and compare intensities only within staining runs with matched imaging and exposure conditions (standard IHC practice). Report separate cell populations where possible, because RNF123 staining varies across annotated tissues and cell types (HPA tissue IHC: positive and negative cell profiles).

How can I distinguish credible RNF123 positivity from staining artefact?

Credible signal should be predominantly cytoplasmic in identifiable cells, consistent with the RNF123 localisation annotations (UniProt Q5XPI4: subcellular location; HPA subcellular: Cytosol). Colon glandular cells offer a high-staining comparator, whereas adipocytes are listed as not detected; interpret either only in its stated cell context (HPA: High in colon glandular cells; HPA: Not detected in adipocytes). Discount edge-heavy staining, necrotic deposits and signal persisting in no-primary controls, and check whether endogenous peroxidase or biotin contributes to DAB development (standard IHC practice). Because tissue staining has medium consistency with RNA expression data, support unexpected cell-specific or nuclear patterns with an independent control before drawing biological conclusions (HPA tissue IHC: Enhanced reliability, medium consistency; standard IHC practice).

Boster reagents

Best RNF123 / E3 ubiquitin-protein ligase RNF123 IHC Antibodies

A09642-1 has paraffin-section IHC images from human intestinal, lung and mammary cancers and mouse intestine (catalog IHC captions); no IF image is supplied (catalog IF image list).

Real IHC data

Anti-RNF123 Antibody ®

Cat # A09642-1

View product →

A09642-1 is listed for IHC and reacts with human, mouse and rat (catalog: applications and reactivity). Its IHC images show paraffin sections of human intestinal, lung and mammary cancers and mouse intestine (catalog IHC captions).

Which to pick: For tissue IHC, choose A09642-1, a rabbit antibody with paraffin-section images and a listed 0.5–1 μg/ml IHC dilution (catalog: host, IHC captions and IHC dilution). No IF/ICC pick is supported: A09642-1 has neither IF/ICC in its application list nor an IF image (catalog: applications and IF image list). For cross-species IHC, A09642-1 lists human, mouse and rat reactivity, but its paraffin-section images cover human and mouse only; the fixative is unreported (catalog: reactivity and IHC captions).

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

References

UniProt Consortium. UniProt entry Q5XPI4 (RN123_HUMAN, E3 ubiquitin-protein ligase RNF123).
Human Protein Atlas. RNF123 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. RNF123 subcellular location (ICC-IF): Mainly localized to the cytosol..
Human Protein Atlas. RNF123 antibody validation summary (2 antibodies).
Identification of Age-associated Proteins and Functional Alterations in Human Retinal Pigment Epithelium. Genomics, proteomics & bioinformatics 2022 — PMC9880895.
Downregulation of the Ubiquitin-E3 Ligase RNF123 Promotes Upregulation of the NF-κB1 Target SerpinE1 in Aggressive Glioblastoma Tumors. Cancers 2020 — PMC7281601.
Combination of artesunate and WNT974 induces KRAS protein degradation by upregulating E3 ligase ANACP2 and β-TrCP in the ubiquitin-proteasome pathway. Cell communication and signaling : CCS 2022 — PMC8934478.
PubMed PMID:15531880 — UniProt-cited evidence.
PubMed PMID:16641997 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.