S100A6 / Protein S100-A6 · IHC design guide

Design Immunohistochemistry for S100A6

Plan chromogenic IHC on paraffin sections using S100A6’s cytoplasmic and nuclear tissue pattern (HPA tissue IHC). The guide covers controls, staining interpretation, and the catalog antibody’s IHC-P conditions; IF/ICC has a separate guide.

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for S100A6 (IHC for S100A6): expected localisation Cytoplasmic and nuclear staining in most tissues (HPA tissue IHC), antibody PB9676, validated IHC image, and IHC protocol steps — Printable **S100A6** IHC protocol sheet — expected localisation Cytoplasmic and nuclear staining in most tissues (HPA tissue IHC), antibody PB9676, controls and protocol steps. Open the full S100A6 IHC guide →

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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 in most tissues (HPA tissue IHC)
	Staining pattern	Most tissues show cytoplasmic and nuclear cell 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	Broad tissue expression limits specificity as a marker (HPA tissue IHC)
	Regulation	No staining-linked regulator is annotated (UniProt)
	Isoform / epitope	No annotated isoforms; one full-length 1–90 chain (UniProt)

Section 1

Recommended S100A6 IHC & IF Protocols

The catalog antibody’s IHC-P protocol (datasheet: PB9676) is accompanied by published rat hippocampus (PMC4013634) and ovarian tumour (PMC12998080) protocols.

Recommended immunohistochemistry (IHC-P) protocol parameters
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 pH 6 retrieval (datasheet: PB9676); the ovarian tumour protocol also used citrate pH 6 (PMC12998080).

Section 2

What Is the Expected S100A6 Staining Pattern?

In paraffin-section IHC, expect cytoplasmic and nuclear S100A6 staining in most tissues, with intensity varying by cell type (HPA tissue IHC: Enhanced reliability; cytoplasmic and nuclear profile). High-staining examples include lung macrophages, liver cholangiocytes and testicular Leydig cells (HPA: High). S100A6 is also assigned to the nuclear envelope and cell membrane and has no transmembrane segment (UniProt P06703: subcellular location and topology).

What am I looking at on my slide?
Distinct cytoplasmic and nuclear staining occurs in lung macrophages or liver cholangiocytes.	These are expected compartments in cell types scored High by HPA (HPA tissue IHC: profile; lung and liver). Judge intensity in the identified cells, not across the entire tissue; neighboring cells need not match their staining (HPA tissue IHC: cell-specific levels).
Staining is exclusively extracellular or confined to a tissue lumen, with no convincing cellular signal.	That distribution does not fit the reported cellular IHC profile (HPA tissue IHC: cytoplasmic and nuclear). Review morphology and detection controls before scoring it as S100A6; UniProt reports no signal peptide or transmembrane segment (UniProt P06703: processing and topology).
Strong staining appears in cardiomyocytes or parathyroid glandular cells.	HPA reports S100A6 as Not detected in those particular cell populations (HPA tissue IHC: heart muscle; parathyroid gland). Check cell identification, antibody specificity and endogenous detection activity; this pattern alone cannot establish which explanation applies (general IHC practice).
Brown signal spreads evenly across cells, connective tissue and empty areas.	A widespread deposit with little cellular contrast is difficult to score as the reported cytoplasmic and nuclear pattern (HPA tissue IHC: profile). Examine no-primary and detection controls, blocking and washes for nonspecific staining or endogenous activity (general IHC practice).
A known High cell population has no visible stain, while the slide's counterstain is intact.	Absence in lung macrophages, for example, conflicts with HPA's High observation for that population (HPA tissue IHC: lung). Confirm that the relevant cells are present, then review antibody and detection controls and the general IHC workflow (general IHC practice).

💡Expected S100A6 appearanceCall a positive result when identifiable High cell populations show clear cytoplasmic, often nuclear, staining (HPA tissue IHC: profile and cell-specific levels); uniform staining of empty or unrelated areas is suspect (general IHC practice).

How each factor affects the staining
Cell choice and internal comparison	HPA scores hematopoietic cells, cerebellar granular cells, cholangiocytes, lung macrophages, rectal endothelial cells, skin fibrohistiocytic cells, splenic red-pulp cells and Leydig cells High (HPA tissue IHC: positive cells). Cardiomyocytes and parathyroid glandular cells are Not detected (HPA tissue IHC: negative cells).
Compartment and membrane appearance	Tissue IHC is described as cytoplasmic and nuclear in most tissues; cerebellar granular cells are specifically marked cytoplasm/membrane (HPA tissue IHC: profile; cerebellum). UniProt also lists the nuclear envelope and cell membrane (UniProt P06703: subcellular location).
Evidence strength and scope	HPA calls the tissue IHC profile Enhanced because staining and RNA expression are highly consistent (HPA tissue IHC: reliability). HPA marks three listed antibodies IHC Enhanced, but that does not identify the cause of an unexpected stain on an individual slide (HPA: antibody validation; general IHC practice).
Protein forms and fixation evidence	UniProt lists a single chain covering residues 1–90, no annotated isoforms, no signal peptide and no propeptide (UniProt P06703: chains, isoforms and processing). Neither supplied source reports S100A6-specific fixation sensitivity; assess preparation effects with routine controls (general IHC practice).
IF/ICC Q&A: what compartment should I expect?	HPA's ICC-IF summary reports plasma membrane and cytosol, with both main locations Enhanced (HPA subcellular ICC-IF). This answers an IF localization question; the tissue IHC interpretation remains cytoplasmic and nuclear (HPA tissue IHC: profile).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
High-reference cells are absent from the section.	The selected field lacks the cells needed for an interpretable comparison (HPA tissue IHC: cell-specific High observations).	Choose a field containing an identified High population, such as lung macrophages or liver cholangiocytes (HPA tissue IHC: lung; liver).
Expected High cells are present but unstained.	The result disagrees with their reported High level; the slide alone does not identify the failed step (HPA tissue IHC: positive cells; general IHC practice).	Check antibody, positive control, chromogenic detection and counterstain, then review the IHC workflow (general IHC practice).
Parathyroid glandular cells or cardiomyocytes stain strongly.	Those populations are reported Not detected; misidentified cells, nonspecific staining or endogenous activity remain possible (HPA tissue IHC: negative cells; general IHC practice).	Recheck morphology and compare no-primary and detection controls before assigning target-specific signal (general IHC practice).
The whole section has diffuse brown background.	Poor cellular contrast can reflect nonspecific binding or endogenous detection activity (general IHC practice).	Inspect no-primary controls and review blocking, washes and chromogen development; score only resolved cellular staining (general IHC practice).
Signal appears only in empty spaces or luminal material.	This does not match the reported cytoplasmic and nuclear tissue pattern (HPA tissue IHC: profile).	Check section morphology and controls, and seek convincing intracellular staining in a reported positive cell type (HPA tissue IHC: positive cells; general IHC practice).
Membrane staining is seen without a clear nuclear component.	Cerebellar granular cells are annotated cytoplasm/membrane, while the overall tissue profile includes nuclear expression (HPA tissue IHC: cerebellum; profile).	Record the tissue and cell type before judging the compartment; compare the observed cells with the matching HPA entry (HPA tissue IHC: cell-specific observations).

Source: UniProt P06703 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing S100A6 staining

IHC analysis of S100A6 using anti-S100A6 antibody (PB9676). S100A6 was detected in paraffin-embedded section of Mouse Testis tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-S100A6 Antibody (PB9676) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

PB9676 · IHCIHC analysis of S100A6 using anti-S100A6 antibody (PB9676). S100A6 was detected in paraffin-embedded section of Mouse Testis tissues. Heat m

IHC analysis of S100A6 using anti-S100A6 antibody (PB9676). S100A6 was detected in paraffin-embedded section of Rat Lung tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-S100A6 Antibody (PB9676) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

PB9676 · IHCIHC analysis of S100A6 using anti-S100A6 antibody (PB9676). S100A6 was detected in paraffin-embedded section of Rat Lung tissues. Heat media

IF analysis of S100A6 using anti-S100A6 antibody (PB9677). S100A6 was detected in an immunocytochemical section of A431 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-S100A6 Antibody (PB9677) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The tissue section was developed using Phalloidin-iFluor 594 Conjugated. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

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.

← 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 strongly (HPA: High in lung macrophages). Use heart muscle as the negative tissue, where cardiomyocytes are listed as not detected (HPA: Not detected in cardiomyocytes); on the lung slide, use neighboring cells without specific chromogenic deposit as internal comparators without assuming a particular nonmacrophage cell type is negative.

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, secondary-only control and a concentration-matched rabbit IgG isotype control for the rabbit primary (selected PB9676 IHC caption: rabbit anti-S100A6), plus S100A6-knockout material as a biological negative if available. In lung, distinguish macrophage pigment from chromogen and quench endogenous peroxidase for chromogenic detection; assess tissue autofluorescence if using IF (HPA: High in lung macrophages; standard IHC/IF practice).

⚠️Feasibility: The selected PB9676 paraffin-section caption reports citrate retrieval at pH 6 for 20 minutes, but does not establish whether retrieval is required (selected PB9676 IHC caption). A target-specific fixation window or fixation effect is unreported, and the caption does not report the fixative (selected PB9676 IHC caption). The supplied evidence does not establish that frozen sections or IF are easier; macrophage pigment can complicate lung chromogenic scoring, and autofluorescence should be checked for IF (HPA: High in lung macrophages; standard IHC/IF practice).

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 →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Heart muscle	Cardiomyocytes	Not detected	Protein (IHC)	HPA →
Parathyroid gland	Glandular cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced S100A6 IHC Tips

Troubleshoot chromogenic S100A6 staining in paraffin sections using the catalog antibody’s tissue image, HPA staining patterns, and S100A6 localisation.

What retrieval conditions should I start with for S100A6 in paraffin sections?

Start with heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet PB9676). Those conditions accompany S100A6 staining in a paraffin-embedded mouse testis section using 1 μg/mL primary antibody overnight at 4°C (datasheet PB9676). If staining is weak, compare a longer or shorter heating interval on adjacent sections while keeping detection and exposure to DAB constant (standard IHC practice). Include a known positive compartment, such as Leydig cells, and inspect morphology before accepting stronger staining as improved retrieval (HPA: High in testis Leydig cells; standard IHC practice).

How should I troubleshoot fixation-related loss of S100A6 staining?

The selected tissue image identifies paraffin embedding but does not state the fixative, so S100A6-specific sensitivity to fixation is unknown (datasheet PB9676). Record the actual fixative and fixation time for each specimen, then compare matched sections processed with the same citrate pH 6 retrieval and detection conditions (standard IHC practice; datasheet PB9676). Check preserved tissue architecture and a positive control before attributing a weak result to fixation (standard IHC practice). Do not infer fixation tolerance from HPA staining patterns or from S100A6’s EF-hand domains, acetylation, or phosphorylation (HPA: tissue IHC; UniProt P06703).

Which cellular compartments should contain credible S100A6 staining?

Assess cytoplasmic and nuclear staining in tissue sections, because both are reported across most tissues (HPA: tissue IHC profile). S100A6 is also annotated at the nuclear envelope and cell membrane, with no transmembrane segment; these annotations support careful compartment review without making membrane-only staining a requirement (UniProt P06703). HPA subcellular imaging identifies enhanced plasma-membrane and cytosolic localisation in cultured cells, which may help frame an apparent peripheral signal (HPA: subcellular). Compare compartments within the expected cell population and against a primary-antibody omission control, especially when DAB obscures fine membrane boundaries (HPA: tissue IHC; standard IHC practice).

Could isoforms or epitope accessibility explain discordant S100A6 staining?

The record lists 0 isoforms and a single 1–90 protein chain, so an isoform switch is not a documented explanation for discordant staining (UniProt P06703). S100A6 contains EF-hand regions at residues 12–47 and 48–83, with annotated modifications at residues 40, 46, and 47 (UniProt P06703). The supplied evidence does not map the catalog antibody’s epitope or show that these modifications change its binding (datasheet PB9676; UniProt P06703). Compare retrieval conditions and controls before assigning a staining difference to epitope masking, calcium binding, or a specific modification (standard IHC practice).

How can IF help check an ambiguous chromogenic S100A6 pattern?

Use IF as a separate validation experiment and multiplex S100A6 with an independently validated marker for the expected cell type, such as Leydig cells in testis (HPA: High in testis Leydig cells; standard IF practice). Select fluorophores after checking the tissue’s autofluorescence, and include single-stain and no-primary controls to distinguish bleed-through and background from S100A6 signal (standard IF practice). For a cytosolic or nuclear epitope, test gentle permeabilisation; for an accessible cell-surface epitope, compare an unpermeabilised condition (HPA: subcellular; standard IF practice). The catalog image documents paraffin-section chromogenic IHC, not an IF fixation or permeabilisation condition for this antibody (datasheet PB9676).

How do I separate widespread S100A6 expression from nonspecific DAB background?

Widespread staining can be plausible because HPA reports cytoplasmic and nuclear S100A6 in most tissues, but uniform DAB haze still requires controls (HPA: tissue IHC profile; standard IHC practice). The catalog image used 10% goat serum blocking, 1 μg/mL primary antibody overnight at 4°C, and a biotin-based DAB detection system (datasheet PB9676). Compare a primary-antibody omission control and, when using biotin-based detection, assess endogenous biotin; block endogenous peroxidase before DAB development (standard IHC practice). Evaluate staining in expected cells alongside cardiomyocytes or parathyroid glandular cells, where HPA reports S100A6 as not detected (HPA: tissue IHC).

How should I score S100A6 across heterogeneous tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring, since HPA reports both cytoplasmic and nuclear tissue expression (HPA: tissue IHC profile; standard IHC practice). Within each annotated region, report the percentage of positive cells and an H-score from 0–300 using intensity categories 0–3 (standard IHC practice). If spatial abundance is the question, report positive cells per mm² of viable tissue rather than raw counts per image (standard IHC practice). Normalise comparisons to the same cell type or viable tissue area, apply one staining threshold across batches, and include a positive control on each run (standard IHC practice).

What distinguishes true S100A6 staining from tissue or detection artefact?

A credible result combines staining in an expected cell population with plausible cytoplasmic or nuclear distribution and an appropriately clean control (HPA: tissue IHC profile; standard IHC practice). High staining in testis Leydig cells or lung macrophages is consistent with HPA, whereas cardiomyocytes are reported as not detected (HPA: tissue IHC). Treat signal confined to section edges, necrotic regions, or an implausible compartment as suspect until serial sections and controls resolve it (standard IHC practice; UniProt P06703 localisation). If brown signal persists when primary antibody is omitted, check endogenous peroxidase and the biotin-based detection workflow before assigning it to S100A6 (standard IHC practice; datasheet PB9676).

Boster reagents

Best S100A6 / Protein S100-A6 IHC Antibodies

PB9676 has documented paraffin-section IHC in mouse, rat and human tissues (PB9676 IHC captions); both antibodies list IHC and IF/ICC applications for these species (catalog applications and reactivity).

Real IHC data

Anti-S100 alpha 6/S100A6 Antibody ®

Cat # PB9676

View product →

PB9676 has IHC images from mouse testis, rat lung, human mammary cancer and human placenta (PB9676 IHC captions); its IF caption names PB9677, so it does not document IF for PB9676 (PB9676 IF caption). M02043 lists IHC and IF/ICC applications with human, mouse and rat reactivity, but has no IHC or IF image captions (M02043 catalog).

Which to pick: Choose PB9676 for paraffin-section IHC because its own captions document that preparation across all three listed species; the fixative is unreported (PB9676 IHC captions). For IF/ICC, both SKUs list the application, but neither has an image caption that documents IF for its own SKU (catalog applications; PB9676 IF caption; M02043 image captions). For cross-species IHC, PB9676 has image evidence in human, mouse and rat tissue, while M02043 is a rabbit monoclonal with listed reactivity in those species but no accompanying images (PB9676 IHC captions; M02043 catalog).

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.