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ORC3 / Origin recognition complex subunit 3 · IHC design guide
Design Immunohistochemistry for ORC3
Plan ORC3 chromogenic IHC around its general nuclear tissue pattern (HPA tissue IHC). Start with the catalog antibody’s human IHC-P dilution range of 1:10–1:50, then assess hematopoietic and glial cell staining with appropriate controls (datasheet: 1:10–1:50; HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ORC3 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 | General nuclear tissue staining; chromosome location annotated (HPA tissue IHC; UniProt) | |
| Staining pattern | Nuclear staining; high in hematopoietic and glial cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Bone marrow+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep formalin fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Marrow peroxidase may confound DAB staining (HPA tissue IHC; standard IHC practice) | |
| Regulation | ORC assembly varies with the cell cycle (UniProt) | |
| Isoform / epitope | 3 isoforms; N-terminal epitope coverage unverified (UniProt; datasheet: N-term) |
Section 1
Recommended ORC3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol appears alongside a published lung adenocarcinoma IHC protocol (PMC9972100).
| Sample | FFPE human lung adenocarcinoma tissue (datasheet A07377-1) |
| Fixation | Image formalin-fixed; duration unreported (datasheet A07377-1); 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-ORC3, 1:10-1:50 (datasheet A07377-1) |
| 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 | ORC3-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: General nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with this page’s Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page retrieval rule); compare citrate pH 6.0 if needed (PMC9972100).
Section 2
What Is the Expected ORC3 Staining Pattern?
ORC3 is expected mainly in nuclei and on chromosomes (UniProt Q9UBD5: subcellular location); it has no transmembrane segment (UniProt Q9UBD5: topology). In paraffin-section IHC, expect nuclear staining across multiple cell types, including hematopoietic cells in bone marrow and glial cells in brain (HPA: general nuclear expression; High in these cells). HPA rates the tissue IHC evidence Enhanced, while reporting medium consistency between staining and RNA expression (HPA: reliability description).
| Distinct nuclear staining in bone-marrow hematopoietic cells or brain glial cells. | This fits ORC3 localisation and the reported High staining in these cells (UniProt Q9UBD5: nucleus; HPA: High in hematopoietic and glial cells). Judge it against morphology and the run controls (general IHC practice). |
| Predominantly cytoplasmic, membranous, or extracellular colour with little nuclear signal. | The dominant compartment conflicts with the reported nuclear pattern (UniProt Q9UBD5: nucleus; HPA: general nuclear expression). Consider nonspecific antibody binding, detection background, or misread pigment (general IHC practice). |
| Strong staining in a cell population reported as Low, while expected nuclei stain weakly. | That distribution merits a specificity check; Low is not absent (HPA: Low in bronchial basal cells, salivary glandular cells, splenic red-pulp cells, and lymph-node germinal-center cells). Cross-reactivity or endogenous detection activity is possible (general IHC practice). |
| Diffuse colour across nuclei, cytoplasm, and surrounding tissue. | A diffuse deposit cannot establish ORC3 localisation (UniProt Q9UBD5: nucleus; HPA: general nuclear expression). Compare a no-primary control and review blocking, washing, and chromogen development (general IHC practice). |
| No nuclear signal in bone-marrow hematopoietic cells or brain glial cells. | These are reported High-staining populations (HPA: tissue IHC). Check the run controls and staining workflow before interpreting the section as biologically negative (general IHC practice); the HPA profile does not guarantee every specimen will stain. |
💡Expected ORC3 appearanceCall a result positive when colour is predominantly nuclear in identifiable cells, especially High-staining bone-marrow hematopoietic cells or brain glia (HPA: tissue IHC; UniProt Q9UBD5: nucleus); strong colour confined to cytoplasm or stroma is suspect (general IHC interpretation).
| Where should the signal be judged? | Use nuclear localisation as the primary readout (UniProt Q9UBD5: nucleus and chromosome; HPA: general nuclear expression). HPA's separate ICC-IF record supports nucleoplasmic localisation (HPA: subcellular). |
| Which tissues provide a useful positive comparison? | Bone-marrow hematopoietic cells and glia in caudate, cerebral cortex, or hippocampus are reported High (HPA: tissue IHC). Adipocytes and listed glandular cells are Medium (HPA: tissue IHC); neither level is a required intensity for every section. |
| How broadly should staining be expected? | HPA describes general nuclear expression and Low tissue specificity at the RNA level (HPA: tissue IHC). Its listed Low-staining cell populations are comparison sites, not validated negative controls (HPA: tissue IHC; no negative entries supplied). |
| How much does antibody validation establish? | HPA053748 is rated Enhanced for IHC, whereas HPA039553 has ICC support without an IHC rating (HPA: antibody validation). The tissue profile has medium staining–RNA consistency (HPA: reliability description); assess a new IHC reagent with controls (general IHC practice). |
| Could biology alter the distribution? | ORC3 belongs to an origin-recognition complex whose assembly changes with cell-cycle stage (UniProt Q9UBD5: subunit). This supports caution when comparing cell populations, but supplies no expected IHC intensity by cell-cycle phase. |
| What should IF/ICC show? | Expect nucleoplasmic signal in the separate IF/ICC assay (HPA: supported nucleoplasmic localisation). That observation helps interpret compartment, but it does not validate an IHC reagent or set a paraffin-section protocol (HPA: antibody validation). |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive comparison tissue is blank. | A failed staining step or weak detection is possible (general IHC practice); bone-marrow hematopoietic cells and brain glia are reported High, not guaranteed positive in every specimen (HPA: tissue IHC). | Check the run control, primary-antibody identity, dilution, retrieval, and detection steps against the reagent's IHC instructions (general IHC practice). |
| Only cytoplasm or membranes stain strongly. | The compartment disagrees with nuclear ORC3 localisation (UniProt Q9UBD5: nucleus; HPA: general nuclear expression); nonspecific binding or background is possible (general IHC practice). | Compare a no-primary control, inspect cell boundaries and nuclei, and reassess the primary and detection conditions (general IHC practice). |
| Background obscures nuclear boundaries. | Excess detection deposit or insufficient blocking or washing may obscure localisation (general IHC practice). | Review blocking and washes, then adjust reagent concentration or chromogen development using controls (general IHC practice). |
| Strong signal appears in HPA Low-staining cells. | Cross-reactivity or endogenous detection activity may contribute (general IHC practice); HPA's Low designation does not mean zero ORC3 (HPA: tissue IHC). | Compare the no-primary control and nuclear localisation, then confirm the result with an independently validated reagent if needed (general IHC practice). |
| Reported Medium tissues stain less than High tissues. | Different reported staining levels are expected: adipocytes and listed glandular cells are Medium, while bone-marrow hematopoietic cells and brain glia are High (HPA: tissue IHC). | Score the cell type and nuclear compartment before changing conditions; compare sections within the same controlled run (general IHC practice). |
| ICC-IF looks nuclear, but paraffin-section IHC is inconclusive. | ICC support and IHC validation are application-specific (HPA: HPA039553 ICC Supported; HPA053748 IHC Enhanced). IF nucleoplasmic localisation alone cannot establish paraffin-section IHC performance. | Use an IHC-validated antibody and judge its staining with positive and no-primary controls (HPA: IHC Enhanced for HPA053748; general IHC practice). |
Experiment evidence
IHC & IF images showing ORC3 staining
A07377-1 · IHCORC3L Antibody (N-term) (A07377-1)immunohistochemistry analysis in formalin fixed and paraffin embedded human lung adenocarcinoma followed b
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000135336-ORC3/tissue/cerebral+cortex#imid_15928701
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000135336-ORC3/tissue/colon#imid_15928630
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000135336-ORC3/tissue/liver#imid_15928615
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000135336-ORC3/tissue/kidney#imid_15928624
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA039553; green = ORC3, blue = nucleus, red = microtubules.
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA039553; green = ORC3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ORC3 IHC & IF
🧪Run bone marrow first and score hematopoietic cells for nuclear staining (HPA: High in hematopoietic cells; UniProt Q9UBD5: nucleus and chromosome). HPA detects ORC3 in all 45 scored tissues, so there is no validated negative tissue; use no-primary and isotype controls, and regard cells on the positive slide as internally negative only when they lack nuclear staining above background (HPA: no negative tissue rows; standard IHC scoring practice).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)
Negative control tissue: None in HPA: ORC3 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ORC3 in HEK293, Rh30, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, an isotype control matched to the primary antibody’s host species and clonality, and a biological specificity control using ORC3 knockout material or peptide competition if the immunizing peptide is available (standard IHC practice). For chromogenic staining of bone marrow, quench endogenous peroxidase and inspect background before scoring DAB-positive nuclei (HPA: bone marrow hematopoietic cells High; standard IHC practice).
⚠️Feasibility: The selected A07377-1 tissue-IHC caption reports formalin-fixed, paraffin-embedded human lung adenocarcinoma with peroxidase-conjugated secondary antibody and DAB, but gives no fixation duration or target-specific fixation window (selected tissue-IHC caption). Retrieval requirements and conditions are unreported in the supplied evidence and require empirical optimization (selected tissue-IHC caption). No supplied evidence establishes that frozen sections or IF/ICC are easier; HPA ICC-IF images support nucleoplasmic localization, while endogenous peroxidase in bone marrow can cause chromogenic background (HPA: Nucleoplasm supported; HPA: bone marrow hematopoietic cells High; standard IHC practice).
HPA tissue IHC evidence for ORC3
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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Caudate | Glial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | High | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: ORC3 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced ORC3 IHC Tips
Use nuclear staining and matched controls to troubleshoot ORC3 chromogenic IHC; assess IF separately with its own controls (UniProt Q9UBD5; HPA tissue IHC).
How should I retrieve ORC3 in paraffin sections when nuclear staining is weak?
Start with Tris-EDTA at pH 9.0, heated to 95–98 °C for 20 min (page retrieval setting). Cool sections in the retrieval buffer before applying the IHC-validated antibody, and process a known positive control alongside the test sections (standard IHC practice; HPA: high staining in bone marrow hematopoietic cells). If nuclei remain weak, compare a shorter heating time or a citrate buffer at pH 6.0 on adjacent sections, keeping antibody concentration and detection constant (standard IHC practice). Score nuclear signal separately from diffuse background because ORC3 is annotated in the nucleus and on chromosomes (UniProt Q9UBD5).
Can fixation explain absent ORC3 staining in formalin-fixed paraffin sections?
The selected A07377-1 image documents ORC3 staining in formalin-fixed, paraffin-embedded human lung adenocarcinoma with peroxidase detection and DAB (A07377-1 tissue-IHC caption). That image establishes a workable specimen preparation, but ORC3-specific sensitivity to fixation duration or delayed fixation is unknown from the supplied evidence (A07377-1 tissue-IHC caption). When staining fails, compare sections with documented fixation histories and hold retrieval at pH 9.0, 95–98 °C for 20 min while changing one variable at a time (page retrieval setting; standard IHC practice). Review nuclear morphology and an independently stained control before attributing lost ORC3 signal to fixation (standard IHC practice; UniProt Q9UBD5: nuclear localisation).
What staining pattern should count as ORC3-positive in tissue IHC?
Score convincing signal within intact nuclei, since ORC3 is annotated in the nucleus and on chromosomes and HPA describes general nuclear tissue expression (UniProt Q9UBD5; HPA tissue IHC). HPA places the supported subcellular signal in the nucleoplasm, so compare DAB with a hematoxylin counterstain before classifying faint cytoplasmic colour as positive (HPA subcellular; standard IHC practice). Bone marrow hematopoietic cells and glial cells in caudate, cerebral cortex and hippocampus have reported high staining, providing tissue context for control selection (HPA tissue IHC). ORC assembly changes across the cell cycle, so heterogeneous nuclear intensity alone does not establish a technical failure (UniProt Q9UBD5).
Could isoforms or epitope accessibility change the ORC3 IHC pattern?
ORC3 has 3 listed isoforms, while the selected A07377-1 tissue-IHC caption describes an N-terminal antibody without defining its recognized residues (UniProt Q9UBD5; A07377-1 tissue-IHC caption). Do not assume that this antibody detects every isoform equally or that its signal identifies a particular isoform without an epitope map and isoform-specific validation (UniProt Q9UBD5; standard antibody validation practice). ORC3 has annotated phosphoserines at residues 23 and 516; their effect on this antibody's IHC staining is unreported (UniProt Q9UBD5; A07377-1 tissue-IHC caption). If nuclear staining varies, compare independently validated antibodies or orthogonal expression data on matched specimens before assigning the difference to splicing or phosphorylation (standard IHC practice).
How should I check ORC3 localisation in a multiplex IF experiment?
Pair ORC3 with a validated marker for the expected cell population, such as a glial-cell marker when examining caudate, and include a nuclear counterstain for cell-level assignment (HPA: high staining in caudate glial cells; standard IF practice). Select a far-red ORC3 fluorophore where tissue autofluorescence is prominent, then inspect single-stain controls before interpreting overlap (standard IF practice). ORC3 has no transmembrane segment and is reported in the nucleoplasm, so use a mild nuclear-access permeabilisation condition and verify that nuclear morphology remains intact (UniProt Q9UBD5 topology; HPA subcellular; standard IF practice). HPA reports ICC/IF images in HEK293, Rh30 and U2OS cells; those images do not establish an IF protocol for the selected IHC antibody (HPA subcellular; A07377-1 tissue-IHC caption).
How can I separate ORC3 staining from chromogenic background?
Compare test sections with a no-primary control and inspect whether brown signal tracks intact nuclei or spreads through stroma, tissue edges and damaged areas (standard IHC practice; UniProt Q9UBD5: nuclear localisation). For peroxidase and DAB detection, include an endogenous peroxidase block and assess residual colour in the no-primary control; these are general workflow steps, not ORC3-specific validation (standard IHC practice). If diffuse colour persists, titrate the antibody and shorten DAB development while holding the pH 9.0 retrieval condition fixed (standard IHC practice; page retrieval setting). Interpret weak nuclear colour against matched positive tissue rather than assuming all DAB deposition represents ORC3 (HPA tissue IHC; standard IHC practice).
What is a defensible way to quantify ORC3 in IHC sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and score only intact nuclei, using either percentage positive or an H-score based on the fractions at intensity levels 0–3 (UniProt Q9UBD5: nuclear localisation; standard IHC scoring practice). Report the number of evaluable nuclei and normalise positive counts to all evaluable nuclei in the same compartment; use cells per mm² only when tissue area is measured (standard IHC scoring practice). Keep retrieval, antibody exposure, DAB development and image settings matched across specimens before comparing scores (standard IHC practice). Because HPA reports medium consistency between staining and RNA expression, treat an IHC score as a protein-staining measurement rather than a direct estimate of transcript abundance (HPA tissue IHC).
Which ORC3-positive patterns are credible, and which suggest artefact?
Credible ORC3 staining is nuclear in viable cells, consistent with its nucleus and chromosome annotation and HPA's general nuclear profile (UniProt Q9UBD5; HPA tissue IHC). High signal in bone marrow hematopoietic cells or glial cells in the listed brain regions is plausible, while an unexpected cell population warrants comparison with morphology and controls (HPA tissue IHC; standard IHC practice). Treat staining confined to section edges, necrotic material or nuclei with poor morphology as suspect, and check a no-primary slide for endogenous peroxidase colour (standard IHC practice). The A07377-1 lung adenocarcinoma image demonstrates DAB staining but does not establish clinical relevance or diagnostic specificity (A07377-1 tissue-IHC caption).
Boster reagents
Best ORC3 / Origin recognition complex subunit 3 IHC Antibodies
The IHC-validated ORC3 antibody has a chromogenic IHC image from formalin-fixed, paraffin-embedded human lung adenocarcinoma (A07377-1 image caption). No IF image is supplied (catalog: IF image list).
Real IHC data
Anti-ORC3L Antibody (N-term)
Cat # A07377-1
A07377-1 is the sole rendered SKU and is listed for human IHC-P (catalog: applications and reactivity). Its own image shows DAB staining of formalin-fixed, paraffin-embedded human lung adenocarcinoma (A07377-1 image caption).
Which to pick: Choose A07377-1 for human paraffin-section IHC: it is rabbit polyclonal, is listed for IHC-P, and has an image from formalin-fixed, paraffin-embedded human lung adenocarcinoma (catalog: host, clonality and applications; A07377-1 image caption). No listed SKU is validated for IF/ICC or has an IF image (catalog: applications and IF image list). No cross-species choice is supported because A07377-1 lists human reactivity only (catalog: reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UBD5 (ORC3_HUMAN, Origin recognition complex subunit 3).
- Human Protein Atlas. ORC3 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ORC3 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. ORC3 antibody validation summary (2 antibodies).
- Systemic analysis of the DNA replication regulator origin recognition complex in lung adenocarcinomas identifies prognostic and expression significance. Cancer medicine 2023 — PMC9972100.
- PubMed PMID:10402192 — UniProt-cited evidence.
- PubMed PMID:11230166 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ORC3 (Origin recognition complex subunit 3). Expected localisation General nuclear tissue staining; chromosome location annotated (HPA tissue IHC; UniProt). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A07377-1 at 1:10-1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: FFPE human lung adenocarcinoma tissue (datasheet A07377-1); 2. Fixation: Image formalin-fixed; duration unreported (datasheet A07377-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-ORC3, 1:10-1:50 (datasheet A07377-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep formalin fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Marrow peroxidase may confound DAB staining (HPA tissue IHC; standard IHC practice); Isoform / epitope: 3 isoforms; N-terminal epitope coverage unverified (UniProt; datasheet: N-term); Regulation: ORC assembly varies with the cell cycle (UniProt). Evidence: UniProt Q9UBD5, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm., Datasheet A07377-1, PMC9972100. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ORC3/immunohistochemistry-ihc-guide.