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S100A6 / Protein S100-A6 · IHC design guide
Design Immunohistochemistry for S100A6
Plan paraffin-section S100A6 IHC around the cytoplasmic and nuclear staining reported in most tissues (HPA tissue IHC). Use high-staining lung macrophages and cardiomyocytes reported as undetected to help assess the staining pattern (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
S100A6 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 and nuclear staining (HPA tissue IHC) | |
| Staining pattern | Most tissues show cytoplasmic and nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet PB9676) | |
| Positive control | Bone marrow+4 more · see all | |
| Negative control | Heart muscle+1 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 | Strong lung macrophage staining can complicate scoring (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | No isoforms or processing; epitopes lie within residues 1–90 (UniProt) |
Section 1
Recommended S100A6 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: PB9676) is accompanied by published S100A6 protocols for rat brain, ovarian tumours, and breast tumours (PMC4013634; PMC12998080; PMC10204293).
| Sample | Paraffin-embedded Mouse Testis tissues; fixative not specified (datasheet PB9676) |
| Fixation | Image fixative and duration unreported (datasheet PB9676); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet PB9676) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9676) |
| Primary antibody | Rabbit anti-S100A6, 0.5-1μg/ml (datasheet PB9676) |
| Primary incubation | Overnight at 4 °C (datasheet PB9676) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9676) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | S100A6-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate retrieval at pH 6 (datasheet: PB9676); the ovarian tumour protocol also used citrate at pH 6 (PMC12998080).
Section 2
What Is the Expected S100A6 Staining Pattern?
S100A6 staining in paraffin sections is expected in the cytoplasm and nucleus of many cell types, with strong signal in selected populations such as lung macrophages and liver cholangiocytes (HPA: tissue IHC, Enhanced reliability). Cell membrane or nuclear envelope staining can also fit its annotated location; S100A6 has no transmembrane segment (UniProt P06703: subcellular location and topology). Judge each compartment against the stained cell type and a matched negative control (general IHC practice).
| Clear cytoplasmic staining, with or without nuclear signal, in lung macrophages or liver cholangiocytes. | This fits the broad tissue profile and two populations scored High (HPA: tissue IHC). Nuclear signal can be genuine, but compare it with the cytoplasmic pattern and cell morphology before scoring (HPA: tissue IHC; general IHC practice). |
| Signal appears only in extracellular material, with no convincing staining of intact cells. | An extracellular-only pattern falls outside the annotated cellular locations (UniProt P06703: subcellular location). Inspect adjacent morphology and the negative control for deposited chromogen or nonspecific staining (general IHC practice). |
| Cardiomyocytes stain strongly while the expected positive population is unstained. | Cardiomyocytes are scored Not detected, whereas several other populations are High (HPA: tissue IHC). Consider nonspecific antibody binding or endogenous detection activity; the pattern alone cannot distinguish them (general IHC practice). |
| Brown signal is widespread, including stroma, empty spaces, and cells expected to be unstained. | This distribution is less persuasive than cell-bound cytoplasmic or nuclear staining (HPA: tissue IHC; general IHC practice). Check the negative control before assigning expression; residual endogenous enzyme activity or nonspecific reagent binding can produce background (general IHC practice). |
| No convincing signal appears in a section containing lung macrophages or liver cholangiocytes. | Both are High populations in the HPA tissue profile (HPA: tissue IHC). First confirm that the relevant cells are present; then assess the run controls and IHC detection steps before calling the specimen negative (general IHC practice). |
💡Expected S100A6 appearanceA convincing positive is cell-bound cytoplasmic and possibly nuclear staining, strong in HPA High populations such as lung macrophages; extracellular-only deposits or strong staining of cardiomyocytes warrant control review (HPA: tissue IHC; UniProt P06703: subcellular location; general IHC practice).
| Cell identity and tissue context | Expression spans most tissues, yet named populations range from High to Not detected (HPA: tissue IHC). Score the relevant cells rather than treating every cell in a section as an equivalent control (general IHC practice). |
| Compartment interpretation | Tissue IHC describes cytoplasmic and nuclear expression; UniProt also annotates the nuclear envelope and cell membrane (HPA: tissue IHC; UniProt P06703: subcellular location). A visible membrane edge alone is insufficient to establish a transmembrane pattern (UniProt P06703: topology). |
| Strength of tissue evidence | HPA rates the tissue IHC profile Enhanced and reports high consistency with RNA expression (HPA: tissue IHC). Its listed IHC antibodies include three with Enhanced status; these ratings support the reported pattern, while each experimental run still needs controls (HPA: antibody validation; general IHC practice). |
| IF/ICC Q&A: where should signal appear? | In ICC-IF, HPA reports enhanced plasma membrane and cytosol localisation (HPA: subcellular ICC-IF). Use that as the answer for cultured-cell images; assess paraffin tissue sections against the tissue IHC profile (HPA: tissue IHC). |
| Processing and isoforms | UniProt lists one chain spanning residues 1–90, no signal peptide or propeptide, and no annotated isoforms (UniProt P06703: processing and isoforms). These annotations provide no basis for predicting a cleaved extracellular staining pattern. |
| Epitope and retrieval limits | The supplied records do not map the catalog antibody epitope or report S100A6-specific fixation sensitivity (UniProt P06703; HPA: tissue IHC and antibody validation). Choose and assess retrieval using the antibody's IHC-P instructions and run controls (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| A known High population has no signal. | The relevant cells may be absent from the section, or the IHC run may have failed; staining level alone cannot identify the cause (HPA: tissue IHC; general IHC practice). | Verify cell identity and morphology, then inspect the positive run control and the antibody's IHC-P preparation and detection steps (general IHC practice). |
| The entire section has a diffuse brown haze. | Nonspecific reagent binding or residual endogenous enzyme activity can obscure cellular staining (general IHC practice). | Compare the negative control, check blocking and wash steps, and assess whether the haze persists where no intact cells are present (general IHC practice). |
| Cardiomyocytes show strong signal. | This conflicts with their Not detected HPA score; nonspecific binding or endogenous detection activity is possible (HPA: heart muscle IHC; general IHC practice). | Review the negative control and detection blocking, then compare staining in an HPA High population from the same run (HPA: tissue IHC; general IHC practice). |
| Staining is confined to extracellular deposits. | An extracellular-only result does not match the annotated cellular locations (UniProt P06703: subcellular location). | Check tissue integrity and the negative control; score only staining clearly associated with intact cells (general IHC practice). |
| A low-scored population stains faintly or inconsistently. | HPA lists low staining in several populations, including skeletal myocytes and hippocampal neuronal cells (HPA: tissue IHC). A faint result needs context rather than automatic classification as failure. | Use a High population and the run controls to judge assay performance; record the faint signal separately from clear High staining (HPA: tissue IHC; general IHC practice). |
| The expected cytoplasmic pattern is hard to distinguish from nuclear signal. | Both compartments can be positive in tissue IHC, so their relative intensity may be difficult to judge on a crowded section (HPA: tissue IHC; general IHC practice). | Use the counterstain and cell boundaries to score each compartment, and avoid calling nuclear-only signal an artefact solely because it differs from neighbouring cells (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing S100A6 staining
PB9676 · IHCIHC analysis of S100A6 using anti-S100A6 antibody (PB9676). S100A6 was detected in paraffin-embedded section of Mouse Testis tissues. Heat m
PB9676 · IHCIHC analysis of S100A6 using anti-S100A6 antibody (PB9676). S100A6 was detected in paraffin-embedded section of Rat Lung tissues. Heat media
PB9676 · IFIF analysis of S100A6 using anti-S100A6 antibody (PB9677). S100A6 was detected in an immunocytochemical section of A431 cells. Enzyme antige
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000197956-S100A6/tissue/cerebral+cortex#imid_834888
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000197956-S100A6/tissue/colon#imid_10273339
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000197956-S100A6/tissue/liver#imid_834831
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000197956-S100A6/tissue/pancreas#imid_834782
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB040549; green = S100A6, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB040549; green = S100A6, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for S100A6 IHC & IF
🧪Run lung first: macrophages should stain (HPA: High in lung macrophages). Use heart muscle as the negative tissue, where cardiomyocytes are not detected (HPA: Not detected in heart muscle cardiomyocytes); on the lung slide, cells without visible staining provide an internal background reference, but their S100A6 status requires independent validation.
Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)
Negative control tissue: Heart muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show S100A6 in A-431, U-251MG, U2OS, with annotated localisation: Plasma membrane (enhanced), Cytosol (enhanced) (HPA subcellular).
Technical controls: Include a no-primary control, a secondary-only control, and a concentration-matched nonimmune rabbit IgG isotype control (PB9676 caption: rabbit primary antibody). Use S100A6 knockout material, if available, as a biological specificity control; block endogenous peroxidase for chromogenic detection and assess macrophage autofluorescence if using IF (HPA: High in lung macrophages).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact PB9676 tissue-IHC caption does not state the fixative (PB9676 caption: fixative unreported). The caption uses citrate retrieval at pH 6 for 20 minutes, but does not establish that retrieval is required (PB9676 caption: citrate retrieval). The supplied evidence does not establish whether frozen sections or IF are easier; lung macrophage signal should be interpreted alongside the chromogenic background or IF autofluorescence controls (HPA: High in lung macrophages).
HPA tissue IHC evidence for S100A6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 → |
| Cerebellum | Granular cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | High | Protein (IHC) | HPA → |
| Lung | Macrophages | High | Protein (IHC) | HPA → |
| Rectum | Endothelial cells | High | Protein (IHC) | HPA → |
Section 3
Advanced S100A6 IHC Tips
Troubleshoot S100A6 staining in paraffin sections by checking retrieval, cell identity, subcellular pattern and controls before comparing chromogenic signal across samples.
How should I retrieve S100A6 when staining is weak in paraffin sections?
Start with heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet PB9676). That condition produced a chromogenic S100A6 image in a paraffin-embedded mouse testis section, although the caption does not identify its fixative (datasheet PB9676). If staining remains weak, compare retrieval times on adjacent sections while keeping antibody concentration, detection and development constant (standard IHC practice). Include a known positive cell population, such as Leydig cells, and a primary-omission control to distinguish retrieval failure from detection background (HPA: High in Leydig cells; standard IHC practice). Record tissue damage alongside signal intensity, since stronger development alone cannot establish improved antigen retrieval (standard IHC practice).
Could fixation explain inconsistent S100A6 staining between paraffin blocks?
S100A6-specific sensitivity to fixation is unknown from the supplied evidence, and the selected paraffin-section caption does not state its fixative (datasheet PB9676). Record fixative, time before fixation, fixation duration and processing history for each block before attributing differences to the antigen (standard IHC practice). Compare sections from blocks with documented processing using the same pH 6 citrate retrieval and 20-minute heating condition (datasheet PB9676; standard IHC practice). Keep antibody incubation and DAB development matched, then review morphology and staining in an expected positive cell population (HPA: High in Leydig cells; standard IHC practice). Differences across blocks alone do not establish a target-specific fixation effect (standard IHC practice).
Is nuclear, cytoplasmic or membrane-associated S100A6 staining plausible?
Evaluate the pattern within identified cells: tissue IHC reports cytoplasmic and nuclear expression across most tissues (HPA: tissue IHC profile). UniProt lists the nuclear envelope, cytoplasm and cell membrane, while subcellular imaging emphasizes plasma membrane and cytosol (UniProt P06703: subcellular location; HPA: enhanced subcellular location). S100A6 has no transmembrane segment, so a sharply continuous membrane outline should be checked against adjacent cells and background before assigning it to S100A6 (UniProt P06703: topology; standard IHC practice). Compare compartment patterns in the same staining run, using the hematoxylin counterstain to locate nuclei (standard IHC practice). Score cytoplasmic, nuclear and apparent membrane signal separately when their proportions differ (standard IHC practice).
Could isoforms or epitope accessibility explain discordant S100A6 staining?
The supplied record lists 0 isoforms and one 1–90 protein chain, so an isoform-specific explanation has no support here (UniProt P06703: isoforms and processing). S100A6 contains EF-hand regions at residues 12–47 and 48–83, with reported modifications at residues 40, 46 and 47 (UniProt P06703: domains and modified residues). The catalog antibody's precise epitope is not supplied, so those features cannot establish whether modification or retrieval masks binding (datasheet PB9676; UniProt P06703). If two antibodies disagree, compare their documented epitopes and staining in adjacent sections under matched retrieval and detection conditions (standard IHC practice). Resolve persistent disagreement with an independent specificity control before assigning biological meaning (standard IHC practice).
How should I check S100A6 by multiplex IF after chromogenic IHC?
Treat IF as a separate assay: the supplied PB9676 evidence documents chromogenic staining of a paraffin-embedded section, with no IF condition stated (datasheet PB9676). Multiplex S100A6 with a validated marker for the cell population under study, then assess overlap within individual cells rather than relying on tissue-level coincidence (standard IF practice). Select fluorophores after inspecting unstained tissue autofluorescence, and include single-stain controls for channel bleed-through (standard IF practice). S100A6 lacks a transmembrane segment, but the antibody epitope's accessibility is unspecified; optimize permeabilisation for the compartment being assessed and the chosen antibody (UniProt P06703: topology; standard IF practice). Compare cytosolic or membrane-associated IF patterns with the reported subcellular locations (HPA: enhanced subcellular location).
What controls help separate S100A6 signal from chromogenic background?
Run a primary-omission control through secondary detection and DAB development to expose background from the detection system (standard IHC practice). In the selected tissue image, detection used a biotinylated secondary antibody, streptavidin–biotin complex and DAB, so evaluate background across that whole sequence (datasheet PB9676). Apply a peroxidase block as a general chromogenic IHC step, and assess whether background persists when the primary antibody is omitted (standard IHC practice). If diffuse signal increases with DAB development time, shorten development consistently across sections and reassess expected cellular compartments (standard IHC practice). Compare suspect staining with a positive population and tissue morphology before changing the primary concentration (HPA: High in Leydig cells; standard IHC practice).
How can I quantify S100A6 staining without confusing cell composition with expression? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring because S100A6 can appear in cytoplasmic and nuclear compartments across tissues (HPA: tissue IHC profile). For a defined population, report percentage of positive cells and an H-score on a 0–300 scale, or count positive cells per mm² when density is the question (standard IHC practice). Normalize positive-cell counts to the relevant cell population or sampled tissue area, and report the denominator explicitly (standard IHC practice). Use matched retrieval, antibody incubation and DAB development for every compared section; the catalog image used pH 6 citrate retrieval for 20 minutes (datasheet PB9676; standard IHC practice). Exclude damaged or necrotic regions by a prespecified rule (standard IHC practice).
When should an apparent S100A6-positive focus be treated as artefact?
Check whether the signal occupies identifiable cells and a plausible compartment: tissue IHC supports cytoplasmic and nuclear expression, while subcellular imaging supports cytosol and plasma membrane (HPA: tissue IHC profile and enhanced subcellular location). A focus restricted to section edges or necrotic tissue warrants review of morphology and adjacent intact regions before interpretation (standard IHC practice). Compare cell identity with context-specific references, such as high signal in lung macrophages or testicular Leydig cells and no detected signal in cardiomyocytes (HPA: tissue IHC). Check the primary-omission control for residual DAB signal attributable to the detection workflow (standard IHC practice). Report uncertain foci separately from scored positive cells (standard IHC practice).
Boster reagents
Best S100A6 / Protein S100-A6 IHC Antibodies
PB9676 has IHC images of paraffin sections from mouse, rat and human tissues; IF/ICC is also catalog listed, though its IF caption names a different SKU (catalog image captions; applications).
Real IHC data
Anti-S100 alpha 6/S100A6 Antibody ®
Cat # PB9676
PB9676 will render with its own IHC figure from paraffin-embedded mouse testis; additional IHC captions show rat lung, human mammary cancer and human placenta (PB9676 IHC captions). PB9676 is listed for IF/ICC, but its supplied IF caption names PB9677, so that image cannot establish PB9676 IF performance (catalog applications; IF caption).
Which to pick: Choose PB9676 for tissue IHC: its own paraffin-section captions cover mouse, rat and human samples, with 1 μg/ml primary antibody after citrate retrieval at pH 6 for 20 minutes; the fixative is unreported (PB9676 IHC captions). For IF/ICC, M02043 is a rabbit monoclonal listed for those applications, but it has no supplied IF image; PB9676 is also listed for IF/ICC, with the caption mismatch noted above (catalog host/clone, applications and image captions). For IHC across species, PB9676 has the stronger direct evidence because its own captions show staining in mouse, rat and human paraffin sections (PB9676 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P06703 (S10A6_HUMAN, Protein S100-A6).
- Human Protein Atlas. S100A6 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. S100A6 subcellular location (ICC-IF): Localized to the plasma membrane and cytosol..
- Human Protein Atlas. S100A6 antibody validation summary (4 antibodies).
- Expression of S100A6 in rat hippocampus after traumatic brain injury due to lateral head acceleration. International journal of molecular sciences 2014 — PMC4013634.
- Increased cytoplasmic and nuclear S100A6 expression is associated with improved prognosis in ovarian cancer. BMC cancer 2026 — PMC12998080.
- S100A6 inhibits MDM2 to suppress breast cancer growth and enhance sensitivity to chemotherapy. Breast cancer research : BCR 2023 — PMC10204293.
- Elevated S100A6 (Calcyclin) enhances tumorigenesis and suppresses CXCL14-induced apoptosis in clear cell renal cell carcinoma. Oncotarget 2015 — PMC4466641.
- PubMed PMID:3755724 — UniProt-cited evidence.
- PubMed PMID:3036810 — UniProt-cited evidence.
- PubMed PMID:2448309 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for S100A6 (Protein S100-A6). Expected localisation Cytoplasmic and nuclear staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet PB9676). Validated IHC antibody: PB9676 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Heart muscle. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded Mouse Testis tissues; fixative not specified (datasheet PB9676); 2. Fixation: Image fixative and duration unreported (datasheet PB9676); 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: Citrate pH 6 , 20 min (datasheet PB9676); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9676); 8. Primary antibody: Rabbit anti-S100A6, 0.5-1μg/ml (datasheet PB9676); 9. Primary incubation: Overnight at 4 °C (datasheet PB9676). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Strong lung macrophage staining can complicate scoring (HPA tissue IHC); Isoform / epitope: No isoforms or processing; epitopes lie within residues 1–90 (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt P06703, HPA tissue IHC · Enhanced, HPA ICC-IF · plasma membrane and cytosol., Datasheet PB9676, PMC4013634, PMC12998080. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/S100A6/immunohistochemistry-ihc-guide.