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SAGE1 / Sarcoma antigen 1 · IHC design guide
Design Immunohistochemistry for SAGE1
Plan SAGE1 chromogenic IHC in paraffin sections using testis as a positive control; HPA shows nuclear staining in preleptotene spermatocytes (HPA tissue IHC). Use the catalog antibody at 2–5 μg/ml (datasheet A15250) and assess nuclear staining with appropriate controls.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SAGE1 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 | Nuclear staining in testis (HPA tissue IHC) | |
| Staining pattern | Selective staining of preleptotene spermatocyte nuclei (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A15250) | |
| Positive control | Testis | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Presumed off-target staining may mislead interpretation (HPA tissue IHC) | |
| Regulation | Normal expression is restricted to testis (UniProt) | |
| Isoform / epitope | No isoforms or processing variants annotated (UniProt) |
Section 1
Recommended SAGE1 IHC & IF Protocols
The catalog antibody protocol uses EDTA retrieval (datasheet: A15250). The published IHC protocols below report SAGE1 staining conditions in human testis and spermatocytic seminoma (PMC4529184; PMC3210831).
| Sample | Paraffin-embedded human laryngeal squamous cell carcinoma tissue; fixative not specified (datasheet A15250) |
| Fixation | Image fixative and duration unreported (datasheet A15250); 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 A15250); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A15250) |
| Primary antibody | Rabbit anti-SAGE1, 2-5 μg/ml (datasheet A15250) |
| Primary incubation | Overnight at 4 °C (datasheet A15250) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A15250) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SAGE1-positive staining in preleptotene spermatocytes of testis (HPA tissue IHC: High). HPA tissue profile: Selective nuclear expression in testis. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA at pH 8.0 for the catalog antibody (datasheet: A15250); use each published protocol’s stated retrieval conditions when reproducing that study (PMC4529184; PMC3210831).
Section 2
What Is the Expected SAGE1 Staining Pattern?
SAGE1 is a nuclear protein with no transmembrane segment (UniProt Q9NXZ1). In normal testis, expect selective nuclear staining in preleptotene spermatocytes, where HPA reports high expression (HPA tissue IHC: High in preleptotene spermatocytes). HPA rates the tissue profile Enhanced because antibody staining agrees with RNA expression; it also notes presumed off-target binding that was disregarded (HPA tissue IHC: Enhanced reliability).
| Strong chromogenic signal is confined mainly to nuclei of preleptotene spermatocytes in testis, with limited staining elsewhere. | This matches the expected positive cell type, compartment and High staining level (HPA tissue IHC: preleptotene spermatocytes, High; UniProt Q9NXZ1: nucleus). Score the target signal by its nuclear distribution in the identified cells, rather than by overall section darkness (general IHC practice). |
| Membranes or cytoplasm stain prominently while the expected spermatocyte nuclei remain faint. | That distribution conflicts with SAGE1's nuclear annotation and lack of a transmembrane segment (UniProt Q9NXZ1: nucleus; no transmembrane segment). Treat it as a localization mismatch and examine staining controls, antibody specificity and slide interpretation before calling it SAGE1 (general IHC practice). |
| Strong staining appears in an unexpected cell type, including adipocytes or bronchial respiratory epithelial cells. | HPA reports SAGE1 as not detected in those sampled cells (HPA tissue IHC: adipocytes and bronchial respiratory epithelial cells, Not detected). Such staining warrants investigation for cross-reactivity or endogenous chromogenic activity; appearance alone cannot identify its cause (general IHC practice). |
| A diffuse haze covers tissue, including areas without the expected nuclear pattern. | Diffuse staining is insufficient evidence of SAGE1 expression because the supported tissue pattern is selective and nuclear (HPA tissue IHC: selective nuclear expression in testis). Review background controls, blocking, washes and detection chemistry before interpreting intensity (general IHC practice). |
| A testis section lacks nuclear signal in identifiable preleptotene spermatocytes. | This conflicts with HPA's High positive reference (HPA tissue IHC: preleptotene spermatocytes, High). Check that the cells are present, then review the antibody, staining run and positive-control result; a blank slide alone does not establish biological absence (general IHC practice). |
💡Expected SAGE1 appearanceCall a result positive when preleptotene spermatocytes show strong nuclear staining in testis (HPA tissue IHC: High; UniProt Q9NXZ1: nucleus); prominent membrane staining or staining in HPA-negative cells is suspect (UniProt Q9NXZ1: no transmembrane segment; HPA tissue IHC: Not detected in sampled negative cells).
| Normal-tissue reference | Testis provides the documented positive tissue, specifically preleptotene spermatocytes (HPA tissue IHC: High). HPA lists several sampled normal cell types as Not detected; use each result for that sampled cell type, not as a universal negative claim (HPA tissue IHC: negative cell entries). |
| Antibody validation | HPA003208 has Enhanced IHC validation, while HPA003033 has Supported IHC validation (HPA antibodies: IHC status). The tissue profile has Enhanced reliability, but HPA notes disregarded presumed off-target binding; discordant staining still merits scrutiny (HPA tissue IHC: reliability description). |
| Protein topology and processing | SAGE1 is annotated as nuclear, without a transmembrane segment, signal peptide or propeptide; its listed chain spans residues 1–904 (UniProt Q9NXZ1: subcellular location, topology and processing). These annotations support a nuclear interpretation but provide no target-specific antigen-retrieval or fixation prediction. |
| IF/ICC: where should fluorescence appear? | HPA reports mainly nucleoplasmic signal, with additional nuclear-body localization in ICC-IF (HPA subcellular: enhanced locations). That cellular localization answers the IF question here; it does not supply an IF protocol or change the testis IHC cell-type reference (HPA tissue IHC: preleptotene spermatocytes). |
| Chromogenic detection background | Endogenous enzyme activity and nonspecific reagent binding can contribute signal in chromogenic IHC; suitable detection controls help distinguish them from antibody-dependent staining (general IHC practice). No supplied source establishes a SAGE1-specific effect of fixation or retrieval. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in the testis positive reference. | The expected cells may be absent from the examined area, or the staining run may have failed; HPA reports High staining in preleptotene spermatocytes (HPA tissue IHC). | Confirm cell identity and review the run's positive and reagent controls, antibody use and chromogenic detection steps (general IHC practice). Do not assign a SAGE1-specific fixation cause from this result. |
| Signal is chiefly cytoplasmic or membranous. | The observed compartment disagrees with the nuclear, non-transmembrane annotation (UniProt Q9NXZ1: nucleus; topology). The image alone cannot distinguish cross-reactivity from staining artefact. | Compare with the expected testis nuclear pattern and inspect antibody and detection controls before scoring cells as positive (HPA tissue IHC: selective nuclear expression; general IHC practice). |
| Adipocytes or bronchial respiratory epithelial cells stain strongly. | Those sampled cells are listed as Not detected (HPA tissue IHC: negative entries). Possible explanations include off-target antibody binding or endogenous detection activity (general IHC practice). | Review the cell identification and run appropriate antibody-omission or detection controls; compare with a testis positive reference (general IHC practice; HPA tissue IHC: preleptotene spermatocytes, High). |
| Diffuse chromogen obscures nuclear boundaries. | Background can arise from nonspecific reagent binding, incomplete blocking or detection activity (general IHC practice); it does not match selective nuclear expression (HPA tissue IHC). | Inspect negative controls, blocking, wash steps and detection conditions, then score only interpretable nuclei (general IHC practice). |
| Two antibodies give different IHC patterns. | Their HPA IHC validation levels differ: HPA003208 is Enhanced and HPA003033 is Supported (HPA antibodies: IHC status). HPA also reports presumed off-target binding in the tissue assessment (HPA tissue IHC). | Compare nuclear staining in the same positive cell type and examine negative cells and controls; treat unexplained discordance as unresolved specificity (HPA tissue IHC; general IHC practice). |
| IF shows nuclear bodies as well as broad nuclear signal. | Additional nuclear-body localization is reported alongside predominant nucleoplasmic localization (HPA subcellular: enhanced locations). | Interpret the IF pattern within nuclei and use its dedicated guide for procedure details; retain preleptotene spermatocytes as the IHC tissue reference (HPA subcellular; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing SAGE1 staining
A15250 · IHCIHC analysis of SAGE1 using anti-SAGE1 antibody (A15250). SAGE1 was detected in a paraffin-embedded section of human laryngeal squamous cell
A15250 · IFIF analysis of SAGE1 using anti-SAGE1 antibody (A15250) and anti-Beta Tubulin antibody (M01857-3). SAGE1 was detected in immunocytochemical
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000181433-SAGE1/tissue/cerebral+cortex#imid_1195688
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000181433-SAGE1/tissue/colon#imid_1196478
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000181433-SAGE1/tissue/liver#imid_1195615
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000181433-SAGE1/tissue/kidney#imid_1195616
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA003033; green = SAGE1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA003033; green = SAGE1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SAGE1 IHC & IF
🧪Run testis first and look for staining in preleptotene spermatocytes (HPA: High in preleptotene spermatocytes); use adipose tissue as the negative tissue, assessing its adipocytes (HPA: Not detected in adipocytes). On the testis slide, use unstained cells outside the positive population as an internal background reference, without assuming every other cell type is SAGE1-negative (HPA: High in preleptotene spermatocytes).
Positive control tissue: Testis (Preleptotene spermatocytes, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show SAGE1 in A-431, K-562, U2OS, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a rabbit species- and isotype-matched control for the rabbit primary (caption: rabbit anti-SAGE1); use SAGE1 knockout material, if available, as a biological negative control (standard IHC practice). Quench endogenous peroxidase and assess background in the testis section before interpreting DAB staining (caption: peroxidase secondary and DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A15250 paraffin-section caption does not state the fixative (caption: fixative not stated). The caption reports heat retrieval in EDTA at pH 8.0, but does not establish that this retrieval is required for SAGE1 staining (caption: heat-mediated EDTA retrieval). There is no supplied comparison showing that frozen sections or IF are easier; score nuclear staining and watch for nonspecific DAB background in testis (UniProt: nucleus; HPA: nucleoplasm and nuclear bodies; standard IHC practice).
HPA tissue IHC evidence for SAGE1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 |
|---|---|---|---|---|
| Testis | Preleptotene spermatocytes | High | 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 | Lymphoid tissue | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SAGE1 IHC Tips
Troubleshoot SAGE1 staining in paraffin sections by checking retrieval, nuclear localisation, control tissues, background and scoring before interpreting a chromogenic signal.
Which retrieval condition should I try first for weak SAGE1 staining in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A15250). The selected tissue image used this condition before overnight incubation at 4°C with 2 μg/ml antibody, so keep those conditions fixed while assessing retrieval (datasheet A15250). Include testis with preleptotene spermatocytes as a positive control, and judge improvement by nuclear signal rather than stronger staining everywhere (HPA: high in preleptotene spermatocytes; UniProt Q9NXZ1: nucleus). If signal remains weak, compare a carefully timed retrieval run with a less aggressive run, watching for tissue damage and increased background (standard IHC practice).
How should I troubleshoot possible fixation effects on SAGE1 IHC?
The selected paraffin-section caption does not state a fixative, and target-specific fixation sensitivity is unknown (datasheet A15250: fixative not stated). Record the specimen fixative and fixation duration, then compare sections processed together under the same EDTA pH 8.0 retrieval and detection conditions (datasheet A15250: retrieval; standard IHC practice). Include testis containing preleptotene spermatocytes to check whether the run can show the expected nuclear pattern (HPA: high in preleptotene spermatocytes; HPA: selective nuclear expression). If a specimen stays negative despite a working control, report its processing history and avoid assigning the result to fixation without a controlled comparison (standard IHC practice).
Where should convincing SAGE1 staining appear in a chromogenic section?
Look for nuclear staining: SAGE1 is annotated in the nucleus, and HPA reports mainly nucleoplasmic localisation with additional nuclear bodies (UniProt Q9NXZ1: nucleus; HPA subcellular: nucleoplasm and nuclear bodies). In testis, assess preleptotene spermatocytes as the specified high-expression cell population rather than treating all cells as equivalent controls (HPA: high in preleptotene spermatocytes). Counterstain lightly enough to distinguish DAB signal from nuclear detail, and compare the same compartment in adjacent negative-control sections (standard IHC practice). Predominantly diffuse cytoplasmic or membrane staining warrants a specificity check before scoring, especially when the nuclear positive control is weak (UniProt Q9NXZ1: nucleus; standard IHC practice).
Could an isoform or modified epitope explain discordant SAGE1 staining?
The supplied record lists a single 1–904 chain and 0 annotated isoforms, so it provides no specific splice variant to invoke for discordant IHC (UniProt Q9NXZ1: processing and isoforms). It lists phosphoserines at positions 45, 64 and 238, but their effect on this antibody’s epitope is unreported (UniProt Q9NXZ1: modified residues; datasheet A15250: epitope not stated). Compare staining after the documented EDTA pH 8.0 retrieval while holding antibody concentration and detection constant (datasheet A15250; standard IHC practice). If discordance persists, seek independent epitope information or an orthogonal assay before attributing it to modification (standard IHC practice).
How can IF help verify a questionable nuclear IHC result?
Use IF as a separate check of nuclear localisation while retaining the documented EDTA pH 8.0 method for this page’s chromogenic IHC comparison (datasheet A15250; UniProt Q9NXZ1: nucleus). Multiplex with a marker identifying the expected cell population, such as a validated preleptotene-spermatocyte marker in testis, and check whether signals occur in the same cells (HPA: high in preleptotene spermatocytes; standard IF practice). Choose a fluorophore channel after inspecting unstained tissue autofluorescence, and include single-label controls when channels overlap (standard IF practice). Because SAGE1 has no annotated transmembrane segment and localises to the nucleus, permeabilise fixed cells or sections to allow antibody access to its nuclear epitope; optimise IF fixation independently (UniProt Q9NXZ1: topology and nucleus; standard IF practice).
What should I change when DAB background obscures SAGE1-positive nuclei?
First inspect a no-primary control to separate secondary-reagent, endogenous-peroxidase and chromogen background from antibody-associated staining (standard IHC practice). The selected image used 10% goat serum block, 2 μg/ml primary antibody overnight at 4°C, and DAB detection (datasheet A15250). Apply a peroxidase block and adjust washing, primary concentration or DAB development one variable at a time while preserving the EDTA pH 8.0 retrieval baseline (standard IHC practice; datasheet A15250: retrieval). Require improved nuclear contrast in the positive control without diffuse colour in HPA-listed negative cell populations (HPA: testis preleptotene spermatocytes high; HPA: listed negative tissues not detected).
How should I score SAGE1 IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define a nuclear-positive threshold using control sections, then score the fraction of positive cells and staining intensity within a prespecified cell population (UniProt Q9NXZ1: nucleus; standard IHC practice). An H-score can combine percentage at each intensity level, while positive-cell density per mm² can describe sparse populations (standard IHC practice). Normalise each result to the number of evaluable cells of the intended type or to viable tissue area, and keep section selection and counterstaining consistent (standard IHC practice). Report compartment and cell type alongside the score, using preleptotene spermatocytes as a relevant positive-control population when testis is available (HPA: high in preleptotene spermatocytes; HPA: selective nuclear expression).
How do I distinguish true SAGE1 positivity from a staining artefact?
A convincing result has nuclear signal in an appropriate cell population, consistent with SAGE1’s nuclear annotation and HPA’s selective nuclear testis staining (UniProt Q9NXZ1: nucleus; HPA: selective nuclear expression). The selected paraffin image shows detection in human laryngeal squamous cell carcinoma, while the supplied record also reports expression in head and neck carcinomas (datasheet A15250; UniProt Q9NXZ1: tissue specificity). Scrutinise cytoplasmic-only staining, unexpected cells, section edges and necrotic areas against a no-primary control before calling a case positive (UniProt Q9NXZ1: nucleus; standard IHC practice). If DAB colour persists without primary antibody, investigate endogenous enzyme activity and repeat with an appropriate peroxidase block (standard IHC practice).
Boster reagents
Best SAGE1 / Sarcoma antigen 1 IHC Antibodies
A15250 has IHC data from human paraffin sections of laryngeal squamous cell carcinoma and IF/ICC data from U2OS cells (A15250 image captions); its listed reactivity is human (A15250 catalog).
Real IHC data
Anti-SAGE1 Antibody ®
Cat # A15250
A15250 is the only SKU shown; its IHC image documents staining of human paraffin-embedded laryngeal squamous cell carcinoma at 2 μg/ml (A15250 IHC caption). Its IF image documents staining of U2OS cells at 5 μg/ml (A15250 IF caption).
Which to pick: For tissue IHC, choose A15250 for human paraffin sections; its image caption reports EDTA retrieval at pH 8.0 and DAB detection (A15250 IHC caption). For IF/ICC, choose A15250, which lists both applications and has a U2OS cell image (A15250 catalog applications; A15250 IF caption). Cross-species reactivity is unreported, as are clonality and the IHC specimen’s fixative (A15250 catalog reactivity and clone field; A15250 IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9NXZ1 (SAGE1_HUMAN, Sarcoma antigen 1).
- Human Protein Atlas. SAGE1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. SAGE1 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the nuclear bodies..
- Human Protein Atlas. SAGE1 antibody validation summary (2 antibodies).
- Lack of ADAM2, CALR3 and SAGE1 Cancer/Testis Antigen Expression in Lung and Breast Cancer. PloS one 2015 — PMC4529184.
- OCT2, SSX and SAGE1 reveal the phenotypic heterogeneity of spermatocytic seminoma reflecting distinct subpopulations of spermatogonia. The Journal of pathology 2011 — PMC3210831.
- Multiple cancer/testis antigens are preferentially expressed in hormone-receptor negative and high-grade breast cancers. PloS one 2011 — PMC3060908.
- PubMed PMID:10919659 — UniProt-cited evidence.
- PubMed PMID:15772651 — UniProt-cited evidence.
- PubMed PMID:18669648 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SAGE1 (Sarcoma antigen 1). Expected localisation Nuclear staining in testis (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A15250). Validated IHC antibody: A15250 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Preleptotene spermatocytes); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human laryngeal squamous cell carcinoma tissue; fixative not specified (datasheet A15250); 2. Fixation: Image fixative and duration unreported (datasheet A15250); 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 A15250); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A15250); 8. Primary antibody: Rabbit anti-SAGE1, 2-5 μg/ml (datasheet A15250); 9. Primary incubation: Overnight at 4 °C (datasheet A15250). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Presumed off-target staining may mislead interpretation (HPA tissue IHC); Isoform / epitope: No isoforms or processing variants annotated (UniProt); Regulation: Normal expression is restricted to testis (UniProt). Evidence: UniProt Q9NXZ1, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A15250, PMC4529184, PMC3210831. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SAGE1/immunohistochemistry-ihc-guide.