RAD18 / E3 ubiquitin-protein ligase RAD18 · IHC design guide

Design Immunohistochemistry for RAD18

Plan chromogenic RAD18 IHC in paraffin sections using 2–5 μg/ml of the catalog antibody (datasheet A01622-2). Use appendix lymphoid tissue as a high-staining reference and score nuclear staining in the relevant cell types (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for RAD18 (IHC for RAD18): expected localisation Selective nuclear staining in tissue (HPA tissue IHC), antibody A01622-2, validated IHC image, and IHC protocol steps — Printable **RAD18** IHC protocol sheet — expected localisation Selective nuclear staining in tissue (HPA tissue IHC), antibody A01622-2, controls and protocol steps. Open the full RAD18 IHC guide →

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RAD18 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	Selective nuclear staining in tissue (HPA tissue IHC)
	Staining pattern	Selected germinal center, testis basal and intestinal nuclei (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A01622-2)
	Positive control ⓘ	Appendix+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 sections. (standard IHC practice; not target-specific)
	Caveat	Antibody staining and RNA show medium consistency (HPA tissue IHC)
	Regulation	DNA damage recruits RAD18 to nuclear foci (UniProt)
	Isoform / epitope	No isoforms annotated; one chain spans 1–495 (UniProt)

Section 1

Recommended RAD18 IHC & IF Protocols

The catalog antibody’s IHC-P protocol (datasheet: A01622-2) is accompanied by three published RAD18 IHC protocols (PMC7251712; PMC6558645; PMC9582675).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human gastric signet ring cell carcinoma tissue; fixative not specified (datasheet A01622-2)
Fixation	Image fixative and duration unreported (datasheet A01622-2); 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 A01622-2); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A01622-2)
Primary antibody	Rabbit anti-RAD18, 2-5 μg/ml (datasheet A01622-2)
Primary incubation	Overnight at 4 °C (datasheet A01622-2)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01622-2)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	RAD18-positive staining in lymphoid tissue of appendix (HPA tissue IHC: High). HPA tissue profile: Selective nuclear expression in selected cell types including lymphoid germinal center cells, basal cells in testis and in the intestine. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA, pH 8.0 (datasheet: A01622-2); the published citrate retrieval conditions provide alternatives (PMC6558645; PMC9582675).

Section 2

What Is the Expected RAD18 Staining Pattern?

RAD18 should appear primarily as nuclear staining in selected cells, including lymphoid germinal center cells and basal cells in testis and intestine (HPA tissue IHC). HPA rates the tissue pattern Supported, with medium consistency between staining and RNA data (HPA tissue IHC). Nuclear localization fits chromatin association; UniProt also records a centrosomal location, so compartment calls need context (UniProt Q9NS91). RAD18 has no transmembrane segment (UniProt Q9NS91 topology).

What am I looking at on my slide?
Distinct nuclear staining in selected cells, with stronger staining in appendix lymphoid tissue or colon glandular cells.	This fits the selective nuclear profile and the High levels recorded for those cell types (HPA tissue IHC). Score the stained cell population and nuclear compartment together; nearby unstained cells do not invalidate a selective result (HPA tissue IHC).
Predominantly cell-surface or extracellular staining, without convincing nuclear staining.	Treat this as a suspect pattern: RAD18 is associated with chromatin and has no transmembrane segment (UniProt Q9NS91). Review the negative control and morphology before assigning positivity. Cytoplasmic staining alone needs more caution because UniProt also records a centrosomal location (UniProt Q9NS91).
Strong staining in adipocytes or lung alveolar type I cells, especially when expected nuclei are unstained.	HPA reports RAD18 as Not detected in those cells (HPA tissue IHC). Consider cross-reactivity or endogenous chromogenic activity, both potential IHC confounders (general IHC practice); the unexpected cell type alone cannot identify the cause.
A broad, faint deposit spans several compartments and obscures nuclear boundaries.	This is insufficient for a RAD18-positive call: the reported tissue pattern is selective and nuclear (HPA tissue IHC). Check a negative control, blocking and detection conditions for nonspecific or endogenous signal (general IHC practice), then judge any remaining discrete nuclear staining.
No nuclear staining in an appendix lymphoid or colon glandular region represented on the section.	Those cell types are reported High by HPA, so review the assay before interpreting a negative result (HPA tissue IHC). Confirm that the expected cells are present, then check antibody and detection performance using the validated IHC conditions (general IHC practice).

💡Expected RAD18 appearanceCall a positive result when staining is predominantly nuclear in the expected cells, potentially High in appendix lymphoid or colon glandular cells; diffuse or membrane-dominant color without nuclear definition is suspect (HPA tissue IHC; UniProt Q9NS91 topology).

How each factor affects the staining
Cell population selected for scoring	HPA describes selective nuclear expression, while reporting High staining in several distinct populations, including cardiomyocytes and urothelial cells (HPA tissue IHC). Identify the actual cells on the section before comparing intensity across tissues.
Negative comparator cells	Adipocytes, liver cholangiocytes and lung alveolar type I cells are listed as Not detected (HPA tissue IHC). They can help flag unexpected staining in the same run, provided the relevant cell type is identifiable on the section.
Antibody-specific validation	IHC status differs among listed RAD18 antibodies: HPA006716 and HPA008752 are Supported, while HPA006724 is Approved (HPA antibody validation). Check the IHC status of the antibody actually used; one antibody's status does not transfer to another.
Compartment and target structure	Nuclear staining agrees with RAD18 chromatin association, and a centrosomal location is also recorded (UniProt Q9NS91). The protein has no transmembrane segment or annotated processing into separate chains (UniProt Q9NS91 topology and processing); a membrane rim is therefore difficult to assign to RAD18.
IF/ICC Q: Where should RAD18 appear?	A: Mainly in the nucleoplasm and nuclear bodies, with nuclear speckles also reported (HPA subcellular ICC-IF). This is localization context for IF/ICC; assess paraffin-section chromogenic results against the tissue IHC pattern (HPA tissue IHC).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Expected cells are present, but the slide has no nuclear signal.	A missed assay step or ineffective detection is possible (general IHC practice); the HPA High designation does not establish the cause of a failed run (HPA tissue IHC).	Verify the IHC-validated antibody and its instructions, then check retrieval, primary-antibody incubation, detection reagents and a working positive control (general IHC practice).
Color appears over most of the section, including negative comparator cells.	Nonspecific binding or endogenous detection activity can produce widespread chromogenic signal (general IHC practice); HPA lists adipocytes as Not detected (HPA tissue IHC).	Compare a negative control, check blocking and detection steps, and adjust the antibody concentration within its validated IHC guidance (general IHC practice).
Staining is strong in an HPA-negative cell type.	Cross-reactivity or endogenous activity is possible; HPA reports no detected RAD18 in adipocytes and liver cholangiocytes (HPA tissue IHC). The stain alone cannot distinguish those causes.	Confirm cell identity and nuclear placement, inspect controls, and compare an independently IHC-validated RAD18 antibody if available (general IHC practice; HPA antibody validation).
Only a sharp membrane-like outline is visible.	That distribution conflicts with the nuclear tissue profile and RAD18's lack of a transmembrane segment (HPA tissue IHC; UniProt Q9NS91 topology).	Check the negative control and detection background; withhold a positive call until discrete staining in expected nuclei is visible (general IHC practice; HPA tissue IHC).
A few nuclei stain within an otherwise pale tissue.	Selective expression can give a mixed field (HPA tissue IHC). A field-level average can hide the relevant population (general IHC practice).	Identify the stained cell type and score nuclear staining in that population; compare it with the corresponding HPA cell-type pattern (HPA tissue IHC).
Two RAD18 antibodies give different staining distributions.	Their IHC validation categories can differ, and HPA rates the overall tissue pattern Supported with medium staining-to-RNA consistency (HPA antibody validation; HPA tissue IHC).	Check each antibody's IHC status, compare compartment and cell-type patterns, and use appropriate positive and negative controls before resolving the discrepancy (HPA antibody validation; general IHC practice).

Source: UniProt Q9NS91 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing RAD18 staining

IHC analysis of RAD18 using anti-RAD18 antibody (A01622-2). RAD18 was detected in a paraffin-embedded section of human gastric signet ring cell carcinoma 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-RAD18 Antibody (A01622-2) 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.

A01622-2 · IHCIHC analysis of RAD18 using anti-RAD18 antibody (A01622-2). RAD18 was detected in a paraffin-embedded section of human gastric signet ring c

IHC analysis of RAD18 using anti-RAD18 antibody (A01622-2). RAD18 was detected in a paraffin-embedded section of squamous metaplasia of human renal pelvis 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-RAD18 Antibody (A01622-2) 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.

A01622-2 · IHCIHC analysis of RAD18 using anti-RAD18 antibody (A01622-2). RAD18 was detected in a paraffin-embedded section of squamous metaplasia of huma

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000070950-RAD18/tissue/cerebral+cortex#imid_2776080

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000070950-RAD18/tissue/colon#imid_2788662

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000070950-RAD18/tissue/liver#imid_2788671

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000070950-RAD18/tissue/kidney#imid_2788664

HPA · ICC-IF PC-3Human Protein Atlas immunofluorescence, PC-3, antibody HPA006724; green = RAD18, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for RAD18 IHC & IF

🧪Run colon first: glandular cells should stain for RAD18 (HPA: High in colon glandular cells). Use liver cholangiocytes as the negative tissue (HPA: Not detected in liver cholangiocytes); on the colon slide, neighboring cells without specific RAD18 signal should show only background staining.

Positive control tissue: Appendix (Lymphoid tissue, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RAD18 in PC-3, U-251MG, U2OS, SK-MEL-30, SiHa, A-431, HeLa BAC 6618, HeLa , with annotated localisation: Nucleoplasm (supported), Nuclear bodies (supported) (HPA subcellular).

Technical controls: Include no-primary (secondary-only) and concentration-matched rabbit IgG isotype controls, plus RAD18-knockout tissue or cells as a biological negative (selected A01622-2 caption: rabbit primary antibody; standard IHC practice). Quench endogenous peroxidase and check for endogenous biotin background in colon sections when using the caption’s biotin-based DAB detection (selected A01622-2 caption: biotinylated secondary, SABC and DAB; standard IHC practice).

⚠️Feasibility: A RAD18-specific fixation window or fixation effect is unreported, and the selected A01622-2 paraffin-section caption does not state the fixative; it therefore does not establish fixation. The caption uses heat retrieval in EDTA at pH 8.0, but whether RAD18 staining depends on that retrieval condition is unreported (selected A01622-2 caption). Frozen-section performance is unreported; IF/ICC offers a way to assess the expected nuclear signal (HPA: supported nucleoplasm and nuclear bodies), while endogenous peroxidase in inflammatory cells can complicate colon DAB interpretation (standard IHC practice). The selected A01622-2 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image A01622-2).

HPA tissue IHC evidence for RAD18

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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
Appendix	Lymphoid tissue	High	Protein (IHC)	HPA →
Colon	Glandular cells	High	Protein (IHC)	HPA →
Heart muscle	Cardiomyocytes	High	Protein (IHC)	HPA →
Rectum	Glandular cells	High	Protein (IHC)	HPA →
Thyroid gland	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 →
Breast	Adipocytes	Not detected	Protein (IHC)	HPA →
Liver	Cholangiocytes	Not detected	Protein (IHC)	HPA →
Lung	Alveolar cells type I	Not detected	Protein (IHC)	HPA →

Section 3

Advanced RAD18 IHC Tips

Troubleshoot RAD18 chromogenic IHC in paraffin sections by checking retrieval, nuclear staining, tissue background and cell type before comparing scores.

Which retrieval conditions should I try first for weak RAD18 staining in paraffin sections?

Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A01622-2). The catalog antibody produced a tissue-IHC image using that condition, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet A01622-2). If nuclear staining is weak, check retrieval temperature and duration across matched sections while holding antibody concentration and detection constant (standard IHC practice). Compare signal in the expected cells with adjacent background, because RAD18 tissue expression is selective and nuclear staining is reported in particular cell populations (HPA tissue IHC). Do not treat stronger staining throughout the section as evidence of better retrieval (standard IHC practice).

Could fixation explain weak or uneven RAD18 staining?

RAD18-specific sensitivity to fixation is unknown from the supplied evidence; the catalog image describes a paraffin section but does not state its fixative (datasheet A01622-2). Record the fixative and fixation duration for each specimen, then compare sections processed together before changing retrieval or antibody concentration (standard IHC practice). Overfixation and underfixation can each compromise morphology or antigen detection in paraffin IHC, so inspect nuclear detail and tissue integrity alongside the DAB signal (standard IHC practice). Keep fixation conditions consistent across specimens intended for comparison (standard IHC practice). Neither tissue staining patterns nor RAD18 topology establishes a preferred fixative here.

Where should convincing RAD18 staining appear, and how should cytoplasmic signal be assessed?

Prioritize staining in intact nuclei: RAD18 is nuclear and associates with chromatin (UniProt Q9NS91), while tissue IHC reports selective nuclear expression (HPA tissue IHC). Supported subcellular locations include the nucleoplasm and nuclear bodies, with additional nuclear speckles (HPA subcellular). RAD18 is also annotated at the cytoskeleton and centrosome, so a discrete nonnuclear signal is biologically possible but requires independent validation in paraffin IHC (UniProt Q9NS91). Check whether DAB follows individual cells rather than cut edges, folds or damaged areas (standard IHC practice). Score nuclear and cytoplasmic staining separately, and document the relevant cell type before interpreting a change in localisation (standard IHC practice).

How should an unknown epitope affect RAD18 antibody validation in IHC?

The supplied RAD18 record lists a 495-amino-acid chain and 0 annotated isoforms, but gives no epitope position for this antibody (UniProt Q9NS91; datasheet A01622-2). Consequently, the observed DAB pattern cannot establish which region the antibody recognizes or whether retrieval exposes it equally in every specimen (standard IHC interpretation). RAD18 has annotated phosphorylation sites and a SAP domain at residues 248–282; their relevance to antibody binding is unknown without epitope mapping (UniProt Q9NS91). Compare the catalog antibody with an independently validated antibody against a distinct mapped region when available (standard IHC practice). Require agreement in cellular localisation and staining pattern before assigning discrepant signals to RAD18 (standard IHC practice).

How can IF help assess a disputed RAD18 IHC staining pattern?

Use IF as an orthogonal localisation check for nuclear RAD18, while judging the paraffin-section DAB result on its own controls (UniProt Q9NS91; standard IHC practice). In a germinal-center specimen, multiplex RAD18 with CD20 to identify B cells within the lymphoid area, then assess RAD18 within those cells (HPA tissue IHC; standard IF practice). Select a fluorophore channel with low measured tissue autofluorescence and include an unstained section to establish that background (standard IF practice). Because RAD18 is intracellular and lacks a transmembrane segment, permeabilise cells for access to its nuclear epitope and compare with a no-primary control (UniProt Q9NS91 topology; standard IF practice). IF colocalisation alone does not validate antibody specificity (standard IF practice).

What should I check when RAD18 DAB staining is widespread or diffuse?

First compare a no-primary section with the stained section to identify signal from detection reagents, endogenous enzyme activity or the tissue itself (standard IHC practice). The catalog image used biotinylated secondary antibody, a streptavidin–biotin complex and DAB; test the detection chain separately if it creates diffuse deposits (datasheet A01622-2; standard IHC practice). Include a peroxidase block for chromogenic detection and assess whether longer blocking or additional washing reduces background without erasing nuclear signal (standard IHC practice). The catalog section received 10% goat serum blocking and 2 μg/ml primary antibody overnight (datasheet A01622-2). Reassess antibody concentration only after identifying whether the background originates before or after primary incubation (standard IHC practice).

How should RAD18 staining be scored across paraffin specimens? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and anatomic region before scoring, because RAD18 staining varies by cell type in tissue IHC (HPA tissue IHC). For nuclear DAB, report percent positive cells and an H-score using intensity categories 0–3, giving a possible range of 0–300 (standard IHC practice). If counting focal positive cells, report their density per mm² of viable tissue as well (standard IHC practice). Normalize comparisons to the same eligible cell population or viable tissue area, using matched staining runs and exposure to chromogen (standard IHC practice). Record cytoplasmic signal separately rather than adding it to the nuclear score (UniProt Q9NS91; standard IHC practice).

How can I distinguish true RAD18 staining from tissue or detection artefact?

A plausible positive signal occupies intact nuclei in an appropriate cell population, consistent with RAD18 nuclear localisation and selective nuclear tissue staining (UniProt Q9NS91; HPA tissue IHC). Interpret staining at section edges, folds and necrotic areas cautiously, especially when it lacks clear cellular boundaries (standard IHC practice). Check the no-primary and peroxidase-block controls when brown deposits appear broadly, because endogenous enzyme or detection background can mimic DAB positivity (standard IHC practice). Compare compartments: RAD18 has a documented centrosomal association, but diffuse cytoplasmic DAB alone does not establish target-specific staining (UniProt Q9NS91; standard IHC interpretation). Corroborate unexpected patterns with an independent antibody or another validated assay before drawing a biological conclusion (standard IHC practice).

Boster reagents

Best RAD18 / E3 ubiquitin-protein ligase RAD18 IHC Antibodies

One anti-RAD18 antibody has real IHC images from human paraffin sections of gastric signet ring cell carcinoma, renal pelvis squamous metaplasia, and tonsil (catalog IHC captions). No IF/ICC images are supplied (catalog payload).

Real IHC data

Anti-RAD18 Antibody ®

Cat # A01622-2

View product →

A01622-2 is listed for IHC and shown on human paraffin sections of gastric signet ring cell carcinoma, renal pelvis squamous metaplasia, and tonsil (catalog applications and IHC captions). Its listed reactivity includes human, mouse, and rat, while the supplied IHC images show human tissue only (catalog reactivity and IHC captions).

Which to pick: Choose A01622-2 for paraffin-section IHC; its captions document EDTA retrieval at pH 8.0 and primary antibody at 2 μg/ml, within the listed 2–5 μg/ml IHC range (A01622-2 IHC captions; catalog dilution). No IF/ICC application or image is supplied, so there is no validated IF/ICC choice here (catalog applications and image list). A01622-2 lists mouse and rat reactivity, but its supplied IHC examples are human; the tissue fixative and antibody clone are unreported (catalog reactivity, IHC captions, and clone field).

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

References

UniProt Consortium. UniProt entry Q9NS91 (RAD18_HUMAN, E3 ubiquitin-protein ligase RAD18).
Human Protein Atlas. RAD18 tissue IHC expression (reliability: Supported).
Human Protein Atlas. RAD18 subcellular location (ICC-IF): Mainly localized to the nucleoplasm and nuclear bodies. In addition localized to the nuclear speckles..
Human Protein Atlas. RAD18 antibody validation summary (5 antibodies).
RAD18 promotes colorectal cancer metastasis by activating the epithelial‑mesenchymal transition pathway. Oncology reports 2020 — PMC7251712.
RAD18 may function as a predictor of response to preoperative concurrent chemoradiotherapy in patients with locally advanced rectal cancer through caspase-9-caspase-3-dependent apoptotic pathway. Cancer medicine 2019 — PMC6558645.
RAD18 confers radioresistance of esophagus squamous cell carcinoma through regulating p-DNA-PKcs. Cancer medicine 2022 — PMC9582675.
RAS-RAF-miR-296-3p signaling axis increases Rad18 expression to augment radioresistance in pancreatic and thyroid cancers. Cancer letters 2024 — PMC11132429.
PubMed PMID:10884424 — UniProt-cited evidence.
PubMed PMID:10908344 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.