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S100A3 / Protein S100-A3 · IHC design guide
Design Immunohistochemistry for S100A3
Plan chromogenic S100A3 IHC in paraffin sections using hair cuticle cells as a positive control (HPA tissue IHC). Assess cytoplasmic staining and optimize the IHC-validated antibody within 1:100–1:300 (datasheet: IHC dilution).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
S100A3 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 in hair cuticle cells (HPA tissue IHC) | |
| Staining pattern | Hair cuticle cells show cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Hair | |
| Negative control | Appendix+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 | Low staining in lung macrophages may complicate scoring (HPA tissue IHC) | |
| Regulation | Regulation not specified (UniProt) | |
| Isoform / epitope | No isoforms; residue 51 can be citrullinated (UniProt) |
Section 1
Recommended S100A3 IHC & IF Protocols
The catalog antibody protocol is accompanied by published S100A3 IHC methods for colorectal cancer and liver tissue (PMC3829748; PMC5450779).
| Sample | Paraffin-embedded human lung carcinoma tissue; fixative not specified (datasheet A07744-1) |
| Fixation | Image fixative and duration unreported (datasheet A07744-1); 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 citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane 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-S100A3, 1:100-1:300 (datasheet A07744-1) |
| 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 | S100A3-positive staining in cells in cuticle of hair (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression mainly in cells of the hair cuticle layer. Also expressed in other tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval specification); optimize against the published conditions for your tissue.
Section 2
What Is the Expected S100A3 Staining Pattern?
S100A3 should appear mainly in the cytoplasm of hair cuticle cells, where HPA reports high staining; UniProt places it in the cytoplasm and reports no transmembrane segment (HPA: High in hair cuticle cells; UniProt P33764: cytoplasm, no transmembrane segment). HPA rates its tissue IHC reliability Enhanced, while describing only medium consistency between staining and RNA expression (HPA: tissue IHC reliability).
| Strong cytoplasmic staining in hair cuticle cells, with recognizable hair structure. | This is the clearest expected positive pattern (HPA: High in hair cuticle cells; HPA: cytoplasmic tissue expression). Judge staining within the cuticle cells rather than treating color across the whole hair section as equivalent evidence (general IHC practice). |
| Predominantly nuclear staining, or a sharp membrane outline without convincing cytoplasmic staining. | This is discordant with the dominant tissue pattern (HPA: cytoplasmic tissue expression; UniProt P33764: cytoplasm). Check morphology and controls before calling it S100A3; HPA's additional plasma membrane location comes from ICC-IF and does not establish a membrane-only tissue IHC pattern (HPA: ICC-IF locations). |
| Strong staining in an HPA-listed negative cell population, such as appendix glandular cells or bone marrow hematopoietic cells. | Treat this as unexpected for those cells (HPA: Not detected in appendix glandular cells and bone marrow hematopoietic cells). Cross-reactivity or endogenous chromogen activity are possibilities, not diagnoses; compare an adjacent section processed without primary antibody (general IHC practice). |
| Weak color spread across tissue, including spaces or several unrelated cell populations, without a defined cuticle-cell pattern. | Diffuse background does not establish S100A3 expression. Review blocking, washes, chromogen development, and the no-primary control (general IHC practice). HPA reports some expression outside hair, so assess specific cell populations rather than requiring every other tissue to be blank (HPA: tissue IHC profile). |
| No detectable staining in a section containing identifiable hair cuticle cells. | An absent signal in the best supported positive site conflicts with the reported high cuticle-cell staining (HPA: High in hair cuticle cells). Confirm that cuticle cells are present, then review antibody dilution, retrieval, detection reagents, and control performance (general IHC practice). |
💡Expected S100A3 appearanceCall the result positive when hair cuticle cells show distinct, predominantly cytoplasmic staining of high relative intensity; isolated nuclear color or diffuse staining outside those cells is insufficient (HPA: High in hair cuticle cells; HPA: cytoplasmic tissue expression).
| Which cells give the strongest tissue reference? | Hair cuticle cells are the clearest positive reference (HPA: High in hair cuticle cells; UniProt P33764: inner endocuticle of hair fibers). The HPA tissue profile also reports expression elsewhere, so a positive call outside hair needs its own cell-level interpretation (HPA: tissue IHC profile). |
| How should low-level and negative examples be used? | HPA lists low staining in thyroid, parathyroid and adrenal glandular cells, respiratory epithelial cells of nasopharynx and bronchus, and lung macrophages (HPA: Low). Its listed negative examples refer to specified cells within tissues, not necessarily every cell in each organ (HPA: Not detected examples). |
| What does antibody validation support? | Both HPA042674 and HPA056140 are rated Enhanced for IHC and ICC (HPA: antibody validation). HPA defines Enhanced IHC as a pattern reproduced by independent antibodies or orthogonal data; its overall tissue assessment still notes medium staining–RNA consistency (HPA: validation and tissue reliability). |
| Do molecular features change the expected compartment? | UniProt describes a cytoplasmic protein without a transmembrane segment or signal peptide, and records a processed chain spanning residues 2–101 (UniProt P33764: location, topology, processing). These annotations support a cytoplasmic expectation; they do not specify a retrieval method or predict fixation sensitivity. |
| Do reported modifications predict staining changes? | UniProt records N-acetylalanine at residue 2 and PAD3-associated citrullination at residue 51 (UniProt P33764: modified residues). Without a mapped antibody epitope or modification-specific validation in the supplied evidence, do not infer that either modification raises or lowers IHC signal. |
| Q: What should an IF/ICC image show? | A: Mainly cytosolic signal, with additional Golgi and plasma membrane localization reported by HPA (HPA: ICC-IF subcellular locations). That cell-image observation can guide interpretation, but it does not supply an IF/ICC protocol or override the predominant cytoplasmic tissue IHC pattern (HPA: tissue IHC profile). |
| Situation | Likely cause | Next action |
|---|---|---|
| Hair is present, but the expected cuticle-cell signal is absent. | The section may lack intact, identifiable cuticle cells, or the IHC detection workflow may have failed; HPA reports high staining specifically in cuticle cells (HPA: High in hair cuticle cells). | Confirm cuticle morphology and a positive control, then check primary dilution, retrieval settings, secondary detection, and chromogen development against the chosen assay instructions (general IHC practice). |
| Color appears chiefly in nuclei or as a crisp membrane-only outline. | That distribution disagrees with the dominant cytoplasmic tissue pattern (HPA: tissue IHC profile; UniProt P33764: cytoplasm). HPA's additional membrane localization was observed in ICC-IF (HPA: ICC-IF locations). | Recheck cell boundaries and counterstain, compare with the no-primary control, and seek a reproducible cytoplasmic signal in hair cuticle cells before interpreting the tissue result (general IHC practice; HPA: High in hair cuticle cells). |
| Unexpected strong color appears in listed negative cells. | The signal conflicts with HPA observations for those particular cells (HPA: Not detected examples). Nonspecific binding or endogenous detection activity could produce misleading chromogenic color (general IHC practice). | Compare adjacent sections with and without primary antibody, review blocking and detection controls, and identify the stained cell type before assigning expression (general IHC practice). |
| The slide shows widespread weak haze. | Background from the staining workflow may obscure the cell pattern (general IHC practice). HPA's report of expression in other tissues does not make diffuse, poorly localized color a specific result (HPA: tissue IHC profile). | Check the no-primary control, washes, blocking, and chromogen development; score discrete cellular staining only after background is controlled (general IHC practice). |
| Low staining in lung macrophages or respiratory epithelial cells seems inconsistent with a hair-focused marker. | Hair cuticle cells are the high-staining reference, while HPA also lists low staining in lung macrophages and respiratory epithelial cells of nasopharynx and bronchus (HPA: High; HPA: Low). | Record intensity and cell identity separately, compare with a positive hair section and a no-primary control, and avoid applying a hair-level intensity threshold to every tissue (general IHC practice; HPA: tissue levels). |
| Two antibodies give different tissue patterns. | Both listed antibodies have Enhanced IHC status, yet the overall HPA tissue assessment reports medium consistency with RNA expression (HPA: antibody validation; HPA: tissue reliability). The supplied record does not identify the reason for a particular disagreement. | Compare matched sections, controls, staining conditions, and cell-level localization; report the disagreement rather than selecting one result solely from its validation label (general IHC practice). |
Experiment evidence
IHC & IF images showing S100A3 staining
A07744-1 · IHCImmunohistochemistry analysis of paraffin-embedded human lung carcinoma tissue, using S100A3 Antibody. The picture on the right is blocked w
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000188015-S100A3/tissue/cerebral+cortex#imid_18139763
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000188015-S100A3/tissue/colon#imid_18139689
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000188015-S100A3/tissue/liver#imid_18139690
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000188015-S100A3/tissue/kidney#imid_18139699
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA042674; green = S100A3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA042674; green = S100A3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for S100A3 IHC & IF
🧪Run hair first: cells in the cuticle should stain strongly (HPA: High in hair cuticle cells). Use appendix glandular cells as a negative tissue (HPA: Not detected in appendix glandular cells); on the hair section, compare cells outside the cuticle with the no-primary control for background staining.
Positive control tissue: Hair (Cells in cuticle, HPA High)
Negative control tissue: Appendix (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show S100A3 in A-431, A-549, U2OS, U-251MG, with annotated localisation: Cytosol (enhanced) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, host- and isotype-matched nonimmune immunoglobulin for the antibody’s clonality, and a biological specificity control such as knockout tissue or peptide competition (A07744-1 tissue-IHC caption: peptide-blocked image). For chromogenic IHC, block endogenous peroxidase and assess hair pigment against the control slide; for IF, check hair autofluorescence with an unstained section (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window and the effect of fixation are unreported in the supplied evidence; the fixative for the paraffin-section image is also unreported (A07744-1 tissue-IHC caption: fixative not stated). Antigen-retrieval dependency is unreported, so optimize retrieval empirically for paraffin sections (standard IHC practice). The evidence does not establish that frozen sections or IF are easier for S100A3; hair pigment can complicate chromogenic interpretation, and hair autofluorescence can complicate IF (standard IHC/IF practice).
HPA tissue IHC evidence for S100A3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. External characterization data supports antibody staining.). 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 |
|---|---|---|---|---|
| Hair | Cells in cuticle | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced S100A3 IHC Tips
Troubleshoot S100A3 staining in paraffin sections by checking cuticle cell localization, section controls, and whether the signal survives specificity checks.
What retrieval conditions should I start with for S100A3 in paraffin sections?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). Allow sections to cool in the buffer before washing, then compare a test section with an otherwise matched section processed without retrieval (standard IHC practice). If staining is weak, adjust heating time in small increments while keeping the buffer and detection conditions fixed, and check whether tissue morphology deteriorates (standard IHC practice). Judge improvement by cytoplasmic staining in hair cuticle cells, where expression is high, rather than by a general rise in background (HPA: High in hair cuticle cells; UniProt P33764: cytoplasm).
Could fixation explain weak or uneven S100A3 staining?
The supplied evidence does not establish how S100A3 staining responds to fixation; the selected paraffin section caption does not state a fixative (A07744-1 tissue IHC caption). Record the fixative, fixation duration, processing history, and section age for every specimen before comparing staining intensity (standard IHC practice). If those variables differ, stain sections together with the same retrieval and detection settings, using hair cuticle cells as a reference where available (standard IHC practice; HPA: High in hair cuticle cells). Treat an association between fixation history and signal as an experimental observation until matched specimens confirm it; do not infer fixation sensitivity from cytoplasmic localization or citrullination (UniProt P33764: cytoplasm; citrulline at residue 51).
Where should convincing S100A3 staining appear?
Look primarily for cytoplasmic signal in cells of the hair cuticle layer, the strongest tissue pattern in the supplied evidence (HPA: High in hair cuticle cells; UniProt P33764: cytoplasm). Map the stain against cell boundaries and a counterstain at high magnification before assigning it to a compartment (standard IHC practice). Additional membrane or Golgi patterns are plausible observations, although those locations come from subcellular IF evidence and require confirmation in tissue sections (HPA subcellular: cytosol, plasma membrane, Golgi apparatus). Diffuse staining across unrelated cells or a predominantly nuclear pattern should trigger a review of controls, morphology, and detection background (UniProt P33764: cytoplasm; standard IHC practice).
How should epitope location and modification affect antibody selection?
S100A3 has no listed isoforms or transmembrane segment, so assess antibody specificity against the documented protein sequence and the antibody’s stated immunogen (UniProt P33764: zero isoforms; no transmembrane segment). The protein contains EF hands at residues 12–47 and 50–85, plus reported acetylation at residue 2 and PAD3 mediated citrullination at residue 51 (UniProt P33764). These annotations identify possible epitope considerations; they do not establish whether a particular antibody distinguishes modified protein (UniProt P33764; standard antibody validation practice). For A07744-1, compare staining with its reported synthesized peptide block, then use tissue localization and independent controls to assess remaining signal (A07744-1 tissue IHC caption; HPA: hair cuticle expression).
How can IF help assess an ambiguous chromogenic S100A3 pattern?
Use IF as a separate localization check and multiplex S100A3 with a marker that identifies the expected hair cuticle cells (HPA: High in hair cuticle cells; standard IF practice). Measure autofluorescence in an unstained section, then choose fluorophores and exposure settings that separate marker and S100A3 signals from that baseline (standard IF practice). Because S100A3 is mainly cytosolic and has no transmembrane segment, assess mild permeabilization for access to intracellular epitopes, using matched permeabilized and untreated sections (HPA subcellular: cytosol; UniProt P33764: no transmembrane segment; standard IF practice). Keep single stain and no primary controls for channel bleed through and background, and compare the resulting cell pattern with chromogenic IHC (standard IF practice).
How do I reduce diffuse stain without losing genuine cuticle signal?
First inspect a no primary control for detection background and a matched stained section for signal restricted to plausible cells (standard IHC practice; HPA: High in hair cuticle cells). For peroxidase detection, check the endogenous peroxidase block and wash steps before changing antibody concentration (standard chromogenic IHC practice). Then compare a small antibody dilution series and review blocking conditions, keeping retrieval, chromogen development, and imaging settings consistent (standard IHC practice). A07744-1 has a reported synthesized peptide blocking image in paraffin embedded human lung carcinoma tissue, which can support a specificity check but does not establish that every stained cell is positive (A07744-1 tissue IHC caption).
What should I score when comparing S100A3 across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population before scoring: hair cuticle cells provide the clearest supplied positive reference, while other tissues may show expression (HPA: High in hair cuticle cells; HPA: also expressed in other tissues). Within the selected compartment, report the percentage of positive cells and an H score using consistent intensity categories; record the number of evaluable cells and sections (standard IHC scoring practice). Normalize counts to the number of relevant cells, or report positive cell density per mm² when area is the sampling unit (standard IHC quantification practice). Apply the same positivity threshold, counterstain review, and exclusion rules for damaged tissue across batches, with controls scored alongside specimens (standard IHC practice).
How can I distinguish true S100A3 staining from artefact?
A convincing pattern is cytoplasmic staining in hair cuticle cells, supported by their high reported expression and S100A3’s cytoplasmic annotation (HPA: High in hair cuticle cells; UniProt P33764: cytoplasm). Check apparent positives against the counterstain and tissue morphology so pigment, folds, necrosis, and section edges do not become scored cells (standard IHC practice). Investigate widespread nuclear signal or staining in unexpected cell populations with a no primary control and, for A07744-1, the reported peptide blocking comparison (UniProt P33764: cytoplasm; A07744-1 tissue IHC caption; standard IHC practice). For peroxidase detection, residual endogenous enzyme activity can mimic chromogen signal, so evaluate the peroxidase block before interpreting diffuse staining (standard chromogenic IHC practice).
Boster reagents
Best S100A3 / Protein S100-A3 IHC Antibodies
A07744-1 has a paraffin-section IHC figure and lists IF as an application; Human, Mouse, and Rat reactivity are listed (catalog IHC image alt; catalog applications/reactivity).
Real IHC data
Anti-S-100A3 Antibody
Cat # A07744-1
A07744-1 will render with an IHC figure from paraffin-embedded human lung carcinoma, including a peptide-blocked comparison (catalog IHC image alt). It lists IHC and IF applications and Human, Mouse, and Rat reactivity; no IF figure is supplied (catalog applications/reactivity/IF image alts).
Which to pick: For tissue IHC, choose A07744-1 based on its paraffin-section figure; the fixative is unreported (catalog IHC image alt). For IF/ICC, A07744-1 lists IF at 1:50, but supplies no IF figure or ICC validation (catalog applications/IF dilution/IF image alts). For cross-species work, A07744-1 lists Human, Mouse, and Rat reactivity; it is rabbit-hosted, with no clone or clonality reported (catalog reactivity/host/clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P33764 (S10A3_HUMAN, Protein S100-A3).
- Human Protein Atlas. S100A3 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. S100A3 subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the plasma membrane and the Golgi apparatus..
- Human Protein Atlas. S100A3 antibody validation summary (2 antibodies).
- Role of S100A3 in human colorectal cancer and the anticancer effect of cantharidinate. Experimental and therapeutic medicine 2013 — PMC3829748.
- Role of S100A3 in human hepatocellular carcinoma and the anticancer effect of sodium cantharidinate. Experimental and therapeutic medicine 2017 — PMC5450779.
- S100A gene family: immune-related prognostic biomarkers and therapeutic targets for low-grade glioma. Aging 2021 — PMC8221329.
- New and Old Genes Associated with Primary and Established Responses to Cisplatin and Topotecan Treatment in Ovarian Cancer Cell Lines. Molecules (Basel, Switzerland) 2017 — PMC6151558.
- PubMed PMID:8341667 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for S100A3 (Protein S100-A3). Expected localisation Cytoplasmic in hair cuticle cells (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A07744-1 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Hair (Cells in cuticle); negative Appendix. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung carcinoma tissue; fixative not specified (datasheet A07744-1); 2. Fixation: Image fixative and duration unreported (datasheet A07744-1); 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 citrate buffer, pH 6.0 , 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-S100A3, 1:100-1:300 (datasheet A07744-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Low staining in lung macrophages may complicate scoring (HPA tissue IHC); Isoform / epitope: No isoforms; residue 51 can be citrullinated (UniProt); Regulation: Regulation not specified (UniProt). Evidence: UniProt P33764, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the plasma…, Datasheet A07744-1, PMC3829748, PMC5450779. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/S100A3/immunohistochemistry-ihc-guide.