RAD54B / DNA repair and recombination protein RAD54B · IHC design guide

Design Immunohistochemistry for RAD54B

Plan RAD54B staining in paraffin sections with the catalog antibody at 1:100–1:300 (datasheet: IHC dilution). Compare the reported cytoplasmic tissue staining with the annotated nucleoplasmic location, accounting for the uncertain tissue IHC reliability (HPA tissue IHC; UniProt).

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

Immunohistochemistry protocol sheet for RAD54B (IHC for RAD54B): expected localisation Cytoplasmic tissue staining (HPA tissue IHC); nucleoplasmic location (UniProt), antibody A06804, validated IHC image, and IHC protocol steps — Printable **RAD54B** IHC protocol sheet — expected localisation Cytoplasmic tissue staining (HPA tissue IHC); nucleoplasmic location (UniProt), antibody A06804, controls and protocol steps. Open the full RAD54B IHC guide →

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RAD54B 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 tissue staining (HPA tissue IHC); nucleoplasmic location (UniProt)
	Staining pattern	Cytoplasmic staining in several tissues, including glandular cells (HPA tissue IHC)
	Antigen retrieval	Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen)
	Positive control ⓘ	Parathyroid gland+4 more · see all
	Negative control ⓘ	Adrenal gland+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	Cytoplasmic IHC differs from the annotated nucleoplasmic location (HPA tissue IHC; UniProt)
	Regulation	Active at DNA double-strand breaks (UniProt)
	Isoform / epitope	2 isoforms; epitope coverage is unspecified (UniProt: isoforms; datasheet: epitope)

Section 1

Recommended RAD54B IHC & IF Protocols

Compare the catalog antibody’s IHC-P protocol with three published RAD54B tissue IHC protocols (PMC12917079; PMC6862887; PMC6072389).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A06804)
Fixation	Image fixative and duration unreported (datasheet A06804); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% normal serum of the secondary host, 30 min, room temperature (standard)
Primary antibody	Rabbit anti-RAD54B, 1:100-1:300 (datasheet A06804)
Primary incubation	Overnight at 4 °C (standard)
Detection	HRP-polymer secondary, DAB chromogen 5–10 min (standard)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	RAD54B-positive staining in glandular cells of parathyroid gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues. No signal in the no-primary control.

💡Decision noteStart with Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page retrieval); citrate pH 6.0 is reported in two published protocols (PMC6862887; PMC6072389).

Section 2

What Is the Expected RAD54B Staining Pattern?

RAD54B is assigned to the nucleus and nucleoplasm and is active at DNA double-strand breaks (UniProt Q9Y620). In tissue IHC, however, HPA reports cytoplasmic staining in several tissues, including strong staining of parathyroid glandular cells, testicular Leydig cells and tonsillar squamous epithelial cells (HPA tissue IHC). Treat that compartment difference cautiously: the tissue IHC assessment is Uncertain, and RAD54B has no transmembrane segment (HPA tissue IHC; UniProt Q9Y620).

What am I looking at on my slide?
Cytoplasmic staining in parathyroid glandular cells, Leydig cells or tonsillar squamous epithelial cells.	This matches the reported High tissue IHC pattern, but is provisional because the IHC assessment is Uncertain (HPA tissue IHC). Record the stained cell type and compartment; intensity alone does not establish that the signal is RAD54B.
Predominantly nuclear staining, or staining apparently confined to a tissue lumen or extracellular space.	Nuclear signal is biologically plausible given nucleoplasmic localisation and should not be dismissed solely because HPA tissue IHC describes cytoplasmic staining (UniProt Q9Y620; HPA tissue IHC). Luminal or extracellular signal falls outside the reported intracellular locations; check morphology and staining controls before interpreting it as RAD54B (HPA tissue IHC; HPA ICC-IF).
Strong staining in adrenal glandular cells, cardiomyocytes or myocytes.	These cell types are reported as Not detected in the sampled tissues (HPA tissue IHC). Check whether the signal follows the intended cells; discordant staining may reflect cross-reactivity or endogenous detection activity (general IHC practice). A single discordant section does not establish the cause.
Broad, diffuse chromogen obscures cell boundaries or appears similarly across most structures.	That distribution cannot support a cell-specific RAD54B call against HPA's varied tissue and cell staining levels (HPA tissue IHC). Compare a no-primary control and assess background from detection reagents or inadequate blocking (general IHC practice).
No detectable signal in parathyroid glandular cells, Leyig cells or tonsillar squamous epithelial cells.	These are reported High in tissue IHC, so a blank result warrants a technical check (HPA tissue IHC). Confirm that the selected cells are present, then review the catalog antibody's IHC-P instructions and detection controls (general IHC practice). HPA's Uncertain reliability limits how definitive any one tissue is as a positive control (HPA tissue IHC).

💡Expected RAD54B appearanceA provisional positive is discernible cytoplasmic chromogen in parathyroid glandular cells, Leydig cells or tonsillar squamous epithelial cells at the reported High level; equally strong, indiscriminate staining of cells reported Not detected is suspect (HPA tissue IHC: Uncertain).

How each factor affects the staining
Tissue and cell selection	HPA reports High staining in parathyroid glandular cells, Leydig cells and tonsillar squamous epithelial cells; appendix and colon glandular cells are Medium, while adrenal glandular cells and muscle cell types are Not detected (HPA tissue IHC). Use the cell type, not the organ name alone, when judging a field.
Compartment evidence	UniProt assigns RAD54B to the nucleoplasm and notes activity at DNA double-strand breaks, whereas HPA tissue IHC describes cytoplasmic staining (UniProt Q9Y620; HPA tissue IHC). Keep the assay and its reliability attached to each observation instead of forcing one compartment call across methods.
IF/ICC Q: Where should signal appear?	A: HPA approves both nucleoplasm and cytosol as ICC-IF locations; images are listed for A-431, U-251MG and U2OS (HPA ICC-IF). This answers localisation only: the tissue IHC staining assessment remains Uncertain (HPA tissue IHC).
Antibody evidence	HPA007087 is listed as IHC Uncertain and ICC Approved (HPA antibodies). The tissue IHC assessment also reports medium consistency between antibody staining and RNA expression data (HPA tissue IHC). These ratings support cautious scoring of a chromogenic result, especially when its cell type or compartment differs from the reported pattern.
Protein forms and topology	UniProt lists two isoforms, a single 1–910 chain, no signal peptide or propeptide, and no transmembrane segment (UniProt Q9Y620). The supplied record gives no antibody epitope, so it cannot establish which isoforms a particular IHC reagent detects or predict an epitope-specific retrieval response.
Chromogenic detection background	Endogenous enzyme activity can mimic chromogen staining, depending on the detection system; appropriate blocking and a no-primary control help assess it (general IHC practice). This is a general workflow consideration, not a RAD54B-specific finding or an explanation proven by HPA.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Expected High-staining cells are blank.	The target cells may be absent from the section, or an IHC step or detection control may have failed; the tissue IHC result itself is Uncertain (HPA tissue IHC; general IHC practice).	Locate the reported cell type on the counterstained section, verify the detection control, and follow the catalog antibody's IHC-P instructions for retrieval and dilution (general IHC practice). Do not infer RAD54B-specific fixation sensitivity from this result.
Staining is strong across reported Not detected cell types.	Cross-reactivity or detection background is possible when adrenal glandular cells or muscle cell types stain strongly despite their HPA calls (HPA tissue IHC; general IHC practice).	Compare matched no-primary and detection controls, examine whether color tracks tissue morphology, and score the discordance explicitly (general IHC practice). A clean control does not by itself prove target specificity.
Nuclear signal appears alongside or instead of cytoplasmic signal.	The sources differ by assay: nucleoplasm is assigned by UniProt and approved in HPA ICC-IF, while tissue IHC reports cytoplasmic expression with Uncertain reliability (UniProt Q9Y620; HPA ICC-IF; HPA tissue IHC).	Record nuclear and cytoplasmic scores separately and compare the stained cell types with HPA tissue IHC (general IHC practice; HPA tissue IHC). Avoid calling nuclear staining artefactual solely from the tissue IHC summary.
Chromogen is diffuse, granular or hard to assign to cells.	Detection background or tissue-associated pigment can complicate chromogenic interpretation (general IHC practice); the appearance alone does not identify its source.	Review the no-primary section, counterstain and cell boundaries; adjust blocking or detection conditions as appropriate for the assay (general IHC practice). Score RAD54B only where signal can be assigned to cells.
A Medium-staining tissue looks weaker than a High-staining tissue.	That ordering is consistent with the HPA levels for appendix or colon glandular cells versus the reported High cell types (HPA tissue IHC). Section-to-section intensity still depends on assay conditions (general IHC practice).	Compare like cell types under matched staining and imaging conditions, then report the observed intensity and distribution (general IHC practice). Do not turn HPA's categorical levels into a quantitative threshold.
An IF/ICC result seems inconsistent with tissue IHC.	HPA approves nucleoplasm and cytosol in ICC-IF but describes cytoplasmic tissue IHC with Uncertain reliability (HPA ICC-IF; HPA tissue IHC). The observations come from different assay contexts.	Describe the compartment and assay for each observation, and assess the tissue IHC slide against its reported cell-specific pattern (HPA ICC-IF; HPA tissue IHC). Use the separate IF/ICC guide for that assay's procedure.

Source: UniProt Q9Y620 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing RAD54B staining

Immunohistochemistry analysis of paraffin-embedded human breast carcinoma, using RAD54B Antibody. The picture on the right is blocked with the synthesized peptide.

A06804 · IHCImmunohistochemistry analysis of paraffin-embedded human breast carcinoma, using RAD54B Antibody. The picture on the right is blocked with t

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000197275-RAD54B/tissue/cerebral+cortex#imid_2187384

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000197275-RAD54B/tissue/colon#imid_2187310

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000197275-RAD54B/tissue/liver#imid_2187327

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000197275-RAD54B/tissue/kidney#imid_2187320

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA007087; green = RAD54B, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for RAD54B IHC & IF

🧪Run parathyroid gland first and score its glandular cells for RAD54B staining (HPA: High in parathyroid glandular cells). Run skeletal muscle myocytes as the negative tissue (HPA: Not detected in skeletal muscle myocytes); on the parathyroid slide, use adjacent non-glandular cells as an internal background comparator and record any staining rather than assuming those cells are negative (HPA: High in glandular cells).

Positive control tissue: Parathyroid gland (Glandular cells, HPA High)

Negative control tissue: Adrenal gland (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RAD54B in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (approved), Cytosol (approved) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and clonality (standard IHC practice); use a RAD54B knockout specimen or a peptide-blocked primary as a target-specific negative control (A06804 tissue-IHC caption: peptide-blocked image). For chromogenic detection, quench endogenous peroxidase and assess endogenous biotin if using biotin-based detection (standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the fixative in the selected paraffin-section caption is unreported (A06804 tissue-IHC caption). Retrieval dependency is unreported; optimize antigen retrieval on the positive tissue and compare localization with the expected nucleoplasmic signal (UniProt Q9Y620: nucleoplasm; standard IHC practice). The supplied evidence does not establish that frozen sections or IF are easier, and no parathyroid-specific artefact is reported; assess tissue background with the control slides (HPA: ICC-IF images in A-431, U-251MG and U2OS; standard IHC practice).

HPA tissue IHC evidence for RAD54B

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — 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
Parathyroid gland	Glandular cells	High	Protein (IHC)	HPA →
Testis	Leydig cells	High	Protein (IHC)	HPA →
Tonsil	Squamous epithelial cells	High	Protein (IHC)	HPA →
Appendix	Glandular cells	Medium	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adrenal gland	Glandular cells	Not detected	Protein (IHC)	HPA →
Heart muscle	Cardiomyocytes	Not detected	Protein (IHC)	HPA →
Skeletal muscle	Myocytes	Not detected	Protein (IHC)	HPA →
Smooth muscle	Smooth muscle cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced RAD54B IHC Tips

Troubleshoot RAD54B chromogenic staining in paraffin sections by comparing matched controls, compartment patterns and tissue morphology.

How should I optimize retrieval when RAD54B nuclear staining is weak?

Start with Tris-EDTA pH 9.0 heat retrieval at 95–98 °C for 20 min (page retrieval setting). If staining is weak, compare matched sections with a shorter heating interval while keeping antibody and detection settings constant (standard IHC practice). Citrate pH 6.0 is a fallback to evaluate only after the page condition, with tissue morphology checked alongside signal (standard IHC practice). Assess nuclear staining because RAD54B is nucleoplasmic and active at DNA double-strand breaks (UniProt Q9Y620). The peptide-blocked breast carcinoma image supports a specificity comparison but reports no retrieval method (caption A06804).

Could fixation explain variable RAD54B staining between paraffin sections?

Target-specific RAD54B sensitivity to fixation is unknown from the supplied evidence; the breast carcinoma caption states paraffin embedding but gives no fixative (caption A06804). Record each specimen’s fixative, fixation duration and processing history before comparing stain intensity across sections (standard IHC practice). For a controlled trial, process matched material in 10% neutral buffered formalin using the same fixation interval and section thickness (standard IHC practice). Keep retrieval at Tris-EDTA pH 9.0, 95–98 °C for 20 min during that comparison (page retrieval setting). Interpret differences as processing associated until matched controls establish reproducibility (standard IHC practice).

How should I assess nuclear and cytoplasmic RAD54B staining?

Score nuclear and cytoplasmic staining separately rather than combining them into one positive fraction (standard IHC practice). RAD54B is assigned to the nucleoplasm and is active at DNA double-strand breaks (UniProt Q9Y620). Approved imaging locations include both nucleoplasm and cytosol, while tissue IHC reports cytoplasmic expression in several tissues with uncertain reliability (HPA subcellular; HPA tissue IHC). Compare the 2 compartments within intact cells, using the same counterstain and scoring threshold across matched sections (standard IHC practice). A cytoplasmic-only pattern warrants review of antibody controls and tissue morphology before assigning RAD54B localisation (HPA tissue IHC; standard IHC practice).

Could epitope placement explain discordant RAD54B staining?

RAD54B has 2 recorded isoforms, so check the catalog antibody’s stated immunogen against each sequence before interpreting discordant staining (UniProt Q9Y620; standard IHC practice). The reference protein spans residues 1–910, with an ATP-binding region at 313–480 and a helicase C-terminal region at 649–810 (UniProt Q9Y620). A phosphoserine is recorded at residue 14; its effect on this antibody’s binding is not established by the supplied evidence (UniProt Q9Y620). Compare serial sections under identical retrieval and detection conditions before attributing differences to isoforms or modification (standard IHC practice). Peptide blocking in breast carcinoma provides an antibody competition control, without identifying which isoform was stained (caption A06804).

How can IF clarify an ambiguous RAD54B IHC pattern?

For a separate IF experiment, pair RAD54B with a marker of the expected cell type, such as a cytokeratin marker when examining epithelial cells (HPA tissue IHC; standard IF practice). Use spectrally separated fluorophores and favor a longer wavelength channel for the weaker signal when tissue autofluorescence is prominent (standard IF practice). Permeabilize fixed cells to expose nucleoplasmic RAD54B, then compare a mild 0.1% detergent condition with a matched control for morphology and cytosolic retention (UniProt Q9Y620; HPA subcellular; standard IF practice). Inspect nucleus and cytosol separately because both are approved imaging locations (HPA subcellular). IF images are listed for 3 cell lines, but the supplied record gives no IF fixation or antibody conditions to transfer into this experiment (HPA subcellular).

What should I change when RAD54B chromogenic staining is diffuse?

Run a no-primary control and compare it with a section stained using the catalog antibody to identify detection background (standard IHC practice). Block endogenous peroxidase before chromogen development, and titrate detection time using matched sections rather than increasing every reagent together (standard IHC practice). If the signal remains diffuse, assess blocking, washes and primary antibody dilution against a reference section, recording the chosen values rather than assuming a RAD54B-specific dilution (standard IHC practice). The catalog image includes a peptide-blocked breast carcinoma comparison, but its fixative and processing details are unreported (caption A06804). Review nuclear and cytosolic signal separately because both locations are documented (HPA subcellular).

How should I quantify RAD54B staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define regions and cell populations before scoring, then keep exposure and chromogen development comparable across the slide set (standard IHC practice). Report the percentage of positive cells and, when intensity is reproducible, an H-score from 0–300 using 0–3 intensity categories (standard IHC practice). Score nuclear and cytoplasmic compartments separately because RAD54B has nucleoplasmic and cytosolic localisation evidence (UniProt Q9Y620; HPA subcellular). Normalize positive counts to the number of evaluable cells in the same defined compartment or region, and exclude necrotic or poorly preserved areas (standard IHC practice). Record tissue type and cell population because HPA tissue IHC shows differing cell-specific staining with uncertain reliability (HPA tissue IHC).

When is a RAD54B-positive cell convincing rather than artefactual?

Favor staining in intact, identifiable cells that repeats across matched sections and exceeds no-primary background (standard IHC practice). Nuclear signal fits RAD54B’s nucleoplasmic assignment, while cytosolic signal also has approved subcellular evidence and needs separate assessment (UniProt Q9Y620; HPA subcellular). Check cell identity against the tissue context: HPA reports high staining in testis Leydig cells but rates its overall tissue IHC reliability uncertain (HPA tissue IHC). Discount staining concentrated at section edges, in necrosis or where endogenous peroxidase persists after blocking (standard IHC practice). Treat peptide-blockable staining as supporting antibody specificity in the pictured breast carcinoma section, without extending that control to every tissue or compartment (caption A06804).

Boster reagents

Best RAD54B / DNA repair and recombination protein RAD54B IHC Antibodies

A06804 has an IHC image from paraffin-embedded human breast carcinoma (catalog image caption). IF is listed as an application, and human and mouse are listed as reactive species (catalog).

Real IHC data Immunohistochemistry analysis of paraffin-embedded human breast carcinoma, using RAD54B Antibody. The picture on the right is blocked with the synthesized peptide.

Anti-Rad54B Antibody

Cat # A06804

View product →

A06804 will render with an IHC image of paraffin-embedded human breast carcinoma and a peptide-blocked comparison (catalog image caption). Its listed applications include IHC and IF, and its listed reactivity covers human and mouse; no IF image is supplied (catalog).

Which to pick: For tissue IHC, choose A06804: its own image caption documents paraffin-embedded human breast carcinoma, but does not report the fixative (catalog image caption). For IF/ICC, A06804 lists IF at 1:50, while ICC validation and an IF image are unreported (catalog: applications, IF dilution, image alts). For cross-species work, A06804 lists human and mouse reactivity; clonality is unreported, and the supplied IHC image documents only human tissue (catalog: reactivity, clone, image caption).

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

References

UniProt Consortium. UniProt entry Q9Y620 (RA54B_HUMAN, DNA repair and recombination protein RAD54B).
Human Protein Atlas. RAD54B tissue IHC expression (reliability: Uncertain).
Human Protein Atlas. RAD54B subcellular location (ICC-IF): Localized to the nucleoplasm and cytosol..
Human Protein Atlas. RAD54B antibody validation summary (1 antibodies).
RAD54B as a biomarker for prognosis and immunotherapy response in bladder cancer. Discover oncology 2026 — PMC12917079.
Knockdown of RAD54B expression reduces cell proliferation and induces apoptosis in lung cancer cells. The Journal of international medical research 2019 — PMC6862887.
Amplification of RAD54B promotes progression of hepatocellular carcinoma via activating the Wnt/β-catenin signaling. Translational oncology 2021 — PMC8167290.
Inhibition of RAD54B suppresses proliferation and promotes apoptosis in hepatoma cells. Oncology reports 2018 — PMC6072389.
PubMed PMID:10362364 — UniProt-cited evidence.
PubMed PMID:16421571 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.