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ODF2 / Outer dense fiber protein 2 · IHC design guide
Design Immunohistochemistry for ODF2
This guide maps expected spermatid cytoplasmic staining and tissue controls for ODF2 IHC (HPA tissue IHC). It covers the catalog antibody’s paraffin-section workflow and interpretation of presumed off-target staining (datasheet A05599-3; HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ODF2 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 | Spermatid cytoplasm (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in spermatids (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A05599-3) | |
| Positive control | Bronchus+4 more · see all | |
| Negative control | Adipose tissue+4 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 | Presumed off-target staining may confound interpretation (HPA tissue IHC) | |
| Regulation | High in late spermatids (HPA tissue IHC) | |
| Isoform / epitope | 10 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended ODF2 IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet A05599-3). One published mouse inner-ear protocol uses chromogenic DAB detection for ODF2 (PMC3368262).
| Sample | Paraffin-embedded rat testis tissue; fixative not specified (datasheet A05599-3) |
| Fixation | Image fixative and duration unreported (datasheet A05599-3); 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 A05599-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A05599-3) |
| Primary antibody | Rabbit anti-ODF2, 2-5 μg/ml (datasheet A05599-3) |
| Primary incubation | Overnight at 4 °C (datasheet A05599-3) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A05599-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ODF2-positive staining in ciliated cells (cilia axoneme) of bronchus (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in spermatids. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet A05599-3). The published automated workflow lists CC1 without retrieval timing or temperature (PMC3368262).
Section 2
What Is the Expected ODF2 Staining Pattern?
ODF2 is a cytoskeletal protein associated with centrosomes, centrioles, cilia and sperm tails; it has no transmembrane segment (UniProt Q5BJF6). In tissue IHC, expect cytoplasmic staining in spermatids and staining of cilia axonemes in ciliated cells (HPA: tissue IHC). HPA rates the tissue profile Enhanced, while noting medium consistency with RNA data and presumed off-target staining that was disregarded (HPA: reliability description).
| Strong cytoplasmic staining in elongated or late spermatids. | This matches a high-staining testis cell population (HPA: High in elongated or late spermatids). UniProt also reports abundant ODF2 in step 2 round spermatid cytoplasm, so spermatid stage and position should be recorded when comparing sections (UniProt Q5BJF6 tissue specificity). |
| Strong, narrow staining along the cilia of ciliated epithelial cells. | Cilia axonemes stain strongly in bronchus, fallopian tube and nasopharynx (HPA: High in ciliated cells, cilia axoneme). Score the ciliary border separately from cell bodies; ODF2 also has cilium and centrosome localizations (UniProt Q5BJF6 subcellular location). |
| Predominantly nuclear staining or broad staining across cells without a ciliary or spermatid pattern. | This does not match the supported tissue pattern or cytoskeletal localization (HPA: tissue IHC; UniProt Q5BJF6 subcellular location). Assess antibody specificity and detection artefacts before treating it as ODF2. HPA itself notes presumed off-target staining in its tissue assessment (HPA: reliability description). |
| Staining in cell types listed as undetected, such as adipocytes in adipose tissue. | HPA reports no detectable staining in those adipocytes (HPA: Not detected in adipose adipocytes). Check whether the signal follows tissue structures or the detection chemistry; unexpected staining could reflect cross-reactivity or endogenous detection activity (general IHC practice). |
| Weak haze across tissue, or no staining in an otherwise suitable testis control. | Diffuse haze is difficult to score as a compartment-specific ODF2 result (HPA: spermatid cytoplasmic profile). An unstained testis control is inconclusive without checking for spermatids and a working detection run; late spermatids are a high-staining population (HPA: High in elongated or late spermatids). |
💡Expected ODF2 appearanceCall a convincing IHC positive when spermatids show strong cytoplasmic staining or ciliated cells show strong axonemal staining (HPA: High in testis spermatids and ciliated-cell cilia); diffuse nuclear or widespread unrelated-cell staining is suspect (HPA: tissue profile; UniProt Q5BJF6 subcellular location).
| Which tissue and cell population is scored | HPA records high staining in late spermatids and ciliated-cell axonemes, medium staining in lung macrophages, and no detection in several specified cell populations (HPA: tissue IHC). Treat these as cell-specific observations, not whole-organ positive or negative labels. |
| Subcellular resolution in paraffin sections | ODF2 associates with centrosomes, mother-centriole appendages, cilia and sperm-tail structures (UniProt Q5BJF6 subcellular location). Fine structures may be hard to resolve in chromogenic sections (general IHC practice); judge the visible ciliary or spermatid pattern with tissue morphology. |
| Antibody validation and scope | HPA001874 has Enhanced IHC status, while HPA048841 has no listed IHC status (HPA: antibodies). The Enhanced tissue profile includes a caution about medium RNA concordance and disregarded presumed off-target staining (HPA: reliability description); validation does not make every stained cell specific. |
| Isoform and modification context | UniProt lists 10 ODF2 isoforms and phosphorylation sites (UniProt Q5BJF6 isoforms and modified residues). The supplied sources do not identify the IHC epitope or show that an isoform or modification changes staining; avoid explaining discordant slides through either mechanism without further evidence. |
| Topology and processing | ODF2 has no transmembrane segment, signal peptide or propeptide in this record (UniProt Q5BJF6 topology and processing). A crisp plasma-membrane outline or secreted-looking deposit therefore lacks support as the expected ODF2 pattern; investigate it before scoring. |
| IF/ICC Q: what localization should be expected? | A: HPA places ODF2 mainly at the primary cilium, centrosome, basal body and connecting piece; vesicle and microtubule assignments are uncertain (HPA: subcellular ICC-IF). These imaging observations aid interpretation but do not supply an IF/ICC protocol (HPA: subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in a testis section expected to be positive. | The sampled area may lack the relevant spermatid stages, or the IHC run may have failed (HPA: High in elongated or late spermatids; general IHC practice). | Confirm spermatid stages on the counterstain, then review the run's positive control, antigen retrieval, primary-antibody step and chromogenic detection (general IHC practice). Do not infer ODF2 absence from this slide alone. |
| Cilia appear negative while adjacent cells show broad cytoplasmic color. | The distribution conflicts with the high axonemal staining reported for bronchus, fallopian tube and nasopharynx (HPA: ciliated cells, cilia axoneme). Background or cross-reactivity is possible (general IHC practice). | Identify intact cilia on the counterstain, compare the staining with a no-primary control, and review blocking and detection conditions (general IHC practice). Score axonemes separately from adjacent cytoplasm. |
| Many nuclei or unrelated tissue compartments stain strongly. | Predominant nuclear or widespread unrelated staining is inconsistent with ODF2's reported cytoskeletal locations and HPA tissue pattern (UniProt Q5BJF6 subcellular location; HPA: tissue IHC). | Check the no-primary control and antibody dilution, then compare with a suitable positive tissue section (general IHC practice). Flag the result as unresolved if the compartment remains discordant. |
| A pale brown haze obscures cell boundaries. | Nonspecific antibody binding or detection background can obscure compartment-specific staining (general IHC practice). Haze alone does not reproduce the reported spermatid or axonemal pattern (HPA: tissue IHC). | Review blocking, washing, primary-antibody concentration and detection time; compare a no-primary control processed in the same run (general IHC practice). |
| Lung macrophages stain, but the expected ciliary signal is unclear. | Macrophages have medium staining in lung, while ciliated-cell axonemes have high staining in the listed airway tissues (HPA: tissue IHC). Macrophage color alone is a limited specificity check given HPA's off-target caution (HPA: reliability description). | Score macrophages and cilia as distinct cell populations, inspect a cilia-rich positive section, and compare the no-primary control (general IHC practice; HPA: High in bronchial ciliated-cell cilia). |
| Two antibodies give different tissue patterns. | HPA lists Enhanced IHC status for HPA001874 but no IHC status for HPA048841; it also reports presumed off-target staining in the tissue assessment (HPA: antibodies; HPA: reliability description). | Record each antibody's identity and its staining by cell type and compartment. Recheck controls and compare the result with the validated spermatid and ciliary patterns before interpreting the difference (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing ODF2 staining
A05599-3 · IHCIHC analysis of ODF2 using anti-ODF2 antibody (A05599-3). ODF2 was detected in a paraffin-embedded section of rat testis tissue. Heat mediat
A05599-3 · IHCIHC analysis of ODF2 using anti-ODF2 antibody (A05599-3). ODF2 was detected in a paraffin-embedded section of human ovarian cancer tissue. H
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000136811-ODF2/tissue/cerebral+cortex#imid_1019028
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000136811-ODF2/tissue/colon#imid_1018970
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000136811-ODF2/tissue/liver#imid_1018963
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000136811-ODF2/tissue/kidney#imid_1018972
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA048841; green = ODF2, blue = nucleus, red = microtubules.
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA048841; green = ODF2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ODF2 IHC & IF
🧪Run testis first: elongated or late spermatids should stain (High; HPA: Testis), consistent with the rat testis IHC example (caption: A05599-3). Use adipose tissue as a negative, with adipocytes expected to remain unstained (HPA: Adipose tissue, Not detected); on the testis slide, assess other cells for background relative to the spermatid signal (HPA: Testis, High in elongated or late spermatids).
Positive control tissue: Bronchus (Ciliated cells (cilia axoneme), HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ODF2 in MCF-7, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), Sperm, NIH 3T3, with annotated localisation: Primary cilium (approved), Centrosome (supported), Basal body (approved), Connecting piece (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a rabbit IgG isotype control matched to the primary antibody (caption: rabbit anti-ODF2); use ODF2 knockout tissue, if available, or a validated immunizing-peptide block as a biological specificity control (standard IHC practice). Block endogenous peroxidase and check for nonspecific DAB deposition on the testis section (caption: peroxidase secondary and DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state its fixative (caption: A05599-3). The demonstrated IHC conditions use heat-mediated retrieval in EDTA at pH 8.0, but the evidence does not establish whether retrieval is required or whether frozen sections are easier (caption: A05599-3). IF/ICC localisation can be assessed at centrosomes, basal bodies and primary cilia (HPA: subcellular), but no comparative ease of IF is reported; in testis, assess spermatid staining against background from other cells (HPA: Testis, High in elongated or late spermatids).
HPA tissue IHC evidence for ODF2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded.). 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 |
|---|---|---|---|---|
| Bronchus | Ciliated cells (cilia axoneme) | High | Protein (IHC) | HPA → |
| Fallopian tube | Ciliated cells (cilia axoneme) | High | Protein (IHC) | HPA → |
| Nasopharynx | Ciliated cells (cilia axoneme) | High | Protein (IHC) | HPA → |
| Testis | Elongated or late spermatids | High | Protein (IHC) | HPA → |
| Lung | Macrophages | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Endocrine cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ODF2 IHC Tips
Troubleshoot chromogenic ODF2 staining in paraffin sections using the catalog antibody’s rat testis image, reported localisation, and matched controls.
Which retrieval condition should I try first for ODF2 in paraffin sections?
Start with heat mediated retrieval in EDTA at pH 8.0 (datasheet A05599-3). The catalog antibody detected ODF2 in a paraffin section of rat testis after that retrieval, followed by 2 μg/mL primary antibody overnight at 4°C (datasheet A05599-3). If staining is weak, adjust heating duration or cooling within your tissue workflow while holding antibody concentration and detection conditions constant (general IHC practice). Compare candidate conditions on adjacent sections and look for spermatid cytoplasmic staining rather than a general rise in tissue colour (HPA: cytoplasmic expression in spermatids; general IHC practice).
How should I assess whether fixation is limiting ODF2 staining?
The selected rat testis caption identifies a paraffin section but does not state its fixative, so target specific fixation sensitivity is unknown (datasheet A05599-3: fixative not stated). Record the fixative and fixation duration for each specimen before comparing staining across blocks (general IHC practice). If a block stains weakly, compare adjacent sections using the stated EDTA pH 8.0 retrieval and the same 2 μg/mL primary incubation (datasheet A05599-3; general IHC practice). Include a concurrently processed testis control and judge staining in spermatids, where cytoplasmic expression is reported (HPA: cytoplasmic expression in spermatids; general IHC practice).
Where should convincing ODF2 staining appear in a tissue section?
In testis, look for cytoplasmic staining in spermatids and consider the sperm tail midpiece and part of the principal piece when those structures are visible (HPA: cytoplasmic expression in spermatids; UniProt Q5BJF6: tissue specificity). ODF2 also localises to centrosomes, mother centriole appendages and cilia, so small focal or projection associated signals can be plausible (UniProt Q5BJF6: subcellular location; HPA: primary cilium, centrosome and basal body). HPA reports high axonemal staining in ciliated cells of bronchus, fallopian tube and nasopharynx (HPA: tissue IHC). Assess morphology at adequate magnification before calling broad nuclear staining positive (general IHC practice; UniProt Q5BJF6: subcellular location).
Could an ODF2 isoform or masked epitope explain uneven staining?
ODF2 has 10 annotated isoforms, so staining from one antibody need not represent every isoform equally (UniProt Q5BJF6: isoforms; general antibody practice). The supplied caption gives the antibody concentration and retrieval, but no epitope coordinates; isoform coverage therefore cannot be assigned from this evidence (datasheet A05599-3; UniProt Q5BJF6: isoforms). ODF2 has annotated phosphorylation sites, including residues 73, 74, 92, 95 and 106, but their effect on this antibody is unreported (UniProt Q5BJF6: modified residues; datasheet A05599-3). First compare matched sections under consistent retrieval and detection conditions; interpret remaining differences alongside cell type and compartment (general IHC practice; HPA: cytoplasmic spermatid expression).
How can I check an IHC staining pattern with multiplex immunofluorescence?
Use IF as a separate validation experiment, since the selected antibody caption documents chromogenic staining of a paraffin rat testis section (datasheet A05599-3). Pair ODF2 with a validated spermatid marker in testis, or a ciliated cell marker in ciliated tissue, and check whether the signals occupy the expected structures (HPA: cytoplasmic spermatids and ciliated cell axonemes; general IF practice). Choose fluorophores after measuring tissue autofluorescence, reserving a spectrally separated channel for the weaker signal (general IF practice). ODF2 has no annotated transmembrane segment and has intracellular centrosomal and cytoskeletal locations, so use controlled permeabilisation when the antibody must access an intracellular epitope (UniProt Q5BJF6: topology and subcellular location; general IF practice).
What should I change if DAB appears throughout the section?
Run a no primary control to assess staining contributed by detection reagents and endogenous peroxidase, then apply a peroxidase block as part of the chromogenic workflow (general IHC practice). The selected testis image used 10% goat serum blocking, 2 μg/mL primary antibody overnight at 4°C, and HRP with DAB detection (datasheet A05599-3). If background persists, titrate primary concentration and shorten chromogen development while checking that expected spermatid staining remains visible (general IHC practice; HPA: cytoplasmic expression in spermatids). Assess tissue edges separately, since localised excess colour can obscure the cellular pattern (general IHC practice).
How should I quantify ODF2 staining across testis sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the region and cell population before scoring, focusing on spermatids when measuring the reported testis pattern (HPA: cytoplasmic expression in spermatids; general IHC practice). Record the percentage of positive spermatids and intensity categories for an H-score, or measure positive signal density per mm² when resolving individual cells is impractical (general IHC practice). Normalise cell based scores to the number of evaluable spermatids and area based scores to evaluable tissue area, excluding folds and damaged regions consistently (general IHC practice). Keep retrieval, imaging, DAB development and scoring thresholds consistent across comparison groups (general IHC practice).
How can I distinguish true ODF2 signal from a staining artefact?
Favour a reproducible spermatid cytoplasmic pattern in testis, or ciliary axonemal staining in the reported ciliated tissues (HPA: tissue IHC). Centrosomal or basal body associated puncta can also fit ODF2 localisation, whereas diffuse nuclear colour lacks support from the supplied location record (UniProt Q5BJF6: subcellular location; HPA: subcellular localisation). Check whether signal tracks tissue edges or necrotic regions, and compare with a no primary control for endogenous enzyme colour (general IHC practice). HPA rates tissue staining as enhanced but notes presumed off target binding, so judge unexpected positive cells with compartment, morphology and controls together (HPA: reliability description; general IHC practice).
Boster reagents
Best ODF2 / Outer dense fiber protein 2 IHC Antibodies
The catalog antibody A05599-3 has real IHC images from paraffin sections of rat testis and human ovarian cancer (catalog IHC image captions); no IF image is supplied (catalog IF image list).
Real IHC data
Anti-ODF2 Antibody ®
Cat # A05599-3
A05599-3 is listed for IHC and reacts with human, mouse, and rat samples (catalog applications and reactivity). Its IHC captions show rat testis and human ovarian cancer paraffin sections (catalog IHC image captions).
Which to pick: Choose A05599-3 for paraffin-section IHC; its rat testis figure uses EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody (A05599-3 IHC figure caption). The catalog lists human, mouse, and rat reactivity, but its IHC captions show rat and human tissue only; the fixative is unreported (catalog reactivity and IHC image captions). No SKU in this payload lists IF/ICC validation, so there is no supported IF/ICC pick here (catalog applications and IF image list).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q5BJF6 (ODFP2_HUMAN, Outer dense fiber protein 2).
- Human Protein Atlas. ODF2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ODF2 subcellular location (ICC-IF): Mainly localized to the centrosome, primary cilium, basal body and connecting piece. In addition localized to vesicles, microtubules, calyx, mid piece and principal piece..
- Human Protein Atlas. ODF2 antibody validation summary (2 antibodies).
- Specific expression of Kcna10, Pxn and Odf2 in the organ of Corti. Gene expression patterns : GEP 2012 — PMC3368262.
- Actl7b deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis. Development (Cambridge, England) 2023 — PMC10652042.
- nNOS regulates ciliated cell polarity, ciliary beat frequency, and directional flow in mouse trachea. Life science alliance 2021 — PMC8008965.
- Ectopic Expression of Cenexin1 S796A Mutant in ODF2(+/-) Knockout Background Causes a Sperm Tail Development Defect. Development & reproduction 2012 — PMC4282242.
- PubMed PMID:10381817 — UniProt-cited evidence.
- PubMed PMID:16966375 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ODF2 (Outer dense fiber protein 2). Expected localisation Spermatid cytoplasm (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A05599-3). Validated IHC antibody: A05599-3 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Ciliated cells (cilia axoneme)); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded rat testis tissue; fixative not specified (datasheet A05599-3); 2. Fixation: Image fixative and duration unreported (datasheet A05599-3); 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 retrieval: EDTA pH 8.0 (datasheet A05599-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A05599-3); 8. Primary antibody: Rabbit anti-ODF2, 2-5 μg/ml (datasheet A05599-3); 9. Primary incubation: Overnight at 4 °C (datasheet A05599-3). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Presumed off-target staining may confound interpretation (HPA tissue IHC); Isoform / epitope: 10 isoforms; epitope coverage is unspecified (UniProt); Regulation: High in late spermatids (HPA tissue IHC). Evidence: UniProt Q5BJF6, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the centrosome, primary cilium, basal body and…, Datasheet A05599-3, PMC3368262. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ODF2/immunohistochemistry-ihc-guide.