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CST6 / Cystatin-M · IHC design guide
Design Immunohistochemistry for CST6
Plan chromogenic IHC on paraffin sections using skin corneal-layer cells as a positive reference (HPA tissue IHC). Start with the catalog antibody at 1:200 (datasheet A07408-1) and assess selective cytoplasmic staining in skin squamous epithelium (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CST6 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 skin squamous epithelium (HPA tissue IHC) | |
| Staining pattern | Selective skin staining; corneal-layer cells high (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A07408-1) | |
| Positive control | Skin+1 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific) | |
| Caveat | Secreted CST6 may appear away from producing cells (UniProt; HPA tissue IHC) | |
| Regulation | Psoriatic skin includes spinous-layer expression (UniProt) | |
| Isoform / epitope | No isoforms; mature chain 29–149 has no membrane-sided epitope (UniProt) |
Section 1
Recommended CST6 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A07408-1) is paired with a published colon cancer tissue protocol (PMC10772051).
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A07408-1) |
| Fixation | Image fixative and duration unreported (datasheet A07408-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A07408-1); 20 min, 95–100 °C (standard) |
| 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-CST6, 1:100-1:300 (datasheet A07408-1) |
| 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 | CST6-positive staining in cells in corneal layer of skin (HPA tissue IHC: High). HPA tissue profile: Selective cytoplasmic expression in squamous epithelium of skin. No signal in the no-primary control. |
💡Decision noteStart with heat retrieval in Tris-EDTA at pH 9.0 for the catalog antibody (datasheet: A07408-1). The published colon tissue protocol used citrate at pH 6.0 (PMC10772051).
Section 2
What Is the Expected CST6 Staining Pattern?
CST6 is secreted and has no transmembrane segment (UniProt Q15828: subcellular location and topology). In paraffin-section IHC, expect selective cytoplasmic staining in skin squamous epithelium, with high staining in cells of the corneal layer (HPA: tissue IHC, Enhanced). UniProt also places CST6 in the stratum granulosum of normal skin and eccrine sweat-gland secretory coils (UniProt Q15828: tissue specificity). Interpret layer differences in light of the secreted protein's potentially different RNA and protein locations (HPA: reliability description).
| Strong, selective skin epithelial staining, including cells in the corneal layer (HPA: Skin, High). | This matches the reported skin pattern (HPA: selective cytoplasmic squamous-epithelial expression). Assess cell type, layer and compartment together; a strong chromogen deposit alone does not establish specificity (general IHC practice). |
| Predominantly nuclear staining in skin epithelium, without the expected cytoplasmic pattern. | Nuclear-only staining conflicts with the tissue IHC profile (HPA: selective cytoplasmic expression). Review background and detection controls before assigning CST6 positivity (general IHC practice). Plasma-membrane signal in ICC-IF is separately reported and should not be judged by this nuclear criterion (HPA: subcellular ICC-IF). |
| Strong staining of adipocytes or adrenal glandular cells. | Those cells were not detected in the reported tissue IHC samples (HPA: Adipose tissue and Adrenal gland, Not detected). Consider antibody cross-reactivity or endogenous detection activity, then compare with a control lacking primary antibody (general IHC practice). |
| Diffuse staining across epithelium and surrounding tissue, with little cell or layer distinction. | That distribution is less persuasive than selective skin epithelial staining (HPA: tissue IHC profile). Secreted CST6 may appear beyond its production site (UniProt Q15828: Secreted; HPA: reliability description), so evaluate whether diffuse signal also appears in detection controls (general IHC practice). |
| No staining in the expected skin region. | Absence in a known-positive comparison section conflicts with high staining reported for cells in the skin corneal layer (HPA: Skin, High). Confirm that the relevant layer is present, then check the catalog antibody's IHC-P conditions and the detection run (general IHC practice). |
💡Expected CST6 appearanceCall a result positive when staining is selective and predominantly cytoplasmic in skin squamous epithelium, with high signal in corneal-layer cells (HPA: tissue IHC, Enhanced); widespread control-matched chromogen or dominant nuclear-only staining is suspect (general IHC practice; HPA: cytoplasmic profile).
| Skin layer and cell selection | HPA reports high staining in corneal-layer cells, while UniProt specifies the stratum granulosum of normal skin and eccrine secretory coils (HPA: Skin, High; UniProt Q15828: tissue specificity). Record which structures are actually present before comparing results. |
| Secreted protein and compartment | CST6 has a signal peptide at residues 1–28, a mature chain at 29–149, and no transmembrane segment (UniProt Q15828: processing and topology). Secretion can complicate assignment of staining to the producing cell (HPA: reliability description); retain the observed tissue compartment in the score. |
| Comparison tissue | Kidney collecting ducts show medium staining, while the listed adipocytes and adrenal glandular cells were not detected (HPA: tissue IHC). Use these as pattern comparisons only; a sampled negative category is not proof that every specimen or condition must be negative. |
| Antibody evidence | Two listed antibodies have Enhanced IHC status, HPA044963 and CAB019316 (HPA: antibody validation). HPA also reports high consistency between staining and RNA data while warning that secreted proteins may show different RNA and protein locations (HPA: reliability description). |
| IF/ICC Q&A: Should membrane staining be expected? | Yes, in the separate ICC-IF record: plasma membrane is the approved main location and cytosol an approved additional location (HPA: subcellular ICC-IF). This does not replace the paraffin-section IHC pattern or provide an IF protocol (HPA: tissue IHC; subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| Skin section appears negative. | The scored section may lack the relevant epidermal layer, or the IHC run may have failed (HPA: Skin, High; general IHC practice). | Verify skin-layer anatomy and compare a known-positive skin section processed in the same run; review the catalog antibody's IHC-P instructions and detection controls (general IHC practice). |
| Strong nuclear-only signal dominates. | This disagrees with the reported cytoplasmic tissue pattern (HPA: tissue IHC profile). Nonspecific staining or a detection artifact is possible (general IHC practice). | Check a no-primary control and reassess the antibody dilution and detection conditions against the catalog IHC-P protocol (general IHC practice). |
| Adipocytes stain as strongly as skin epithelium. | Adipocytes were not detected in the HPA tissue panel (HPA: Adipose tissue, Not detected); cross-reactivity or endogenous detection activity may contribute (general IHC practice). | Compare negative tissue and no-primary controls in the same detection run; score CST6 only where the cell and compartment pattern remains credible (general IHC practice; HPA: skin profile). |
| Broad chromogen haze obscures epidermal layers. | Diffuse background can hide the selective epithelial pattern (HPA: tissue IHC profile; general IHC practice). | Inspect a no-primary control, then review blocking, antibody dilution, washing and detection timing under the chosen IHC workflow (general IHC practice). |
| Kidney collecting ducts stain, but less than skin. | That relative result fits the reported medium collecting-duct and high corneal-layer levels (HPA: Kidney, Medium; Skin, High). | Record each tissue and cell type separately; use the skin pattern as the stronger positive comparison when assessing run performance (HPA: tissue IHC; general IHC practice). |
| Staining extends beyond the cells expected to produce CST6. | CST6 is secreted, and HPA cautions that RNA and protein tissue locations may differ (UniProt Q15828: Secreted; HPA: reliability description). | Describe the observed distribution without assigning its source from chromogen alone; compare morphology, the selective skin pattern and detection controls (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing CST6 staining
A07408-1 · IHCImmunohistochemical analysis of paraffin-embedded human tonsil. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDTA, pH9.0 was use
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000175315-CST6/tissue/cerebral+cortex#imid_17205955
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000175315-CST6/tissue/colon#imid_17205873
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000175315-CST6/tissue/liver#imid_17205874
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000175315-CST6/tissue/kidney#imid_17205875
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB019316; green = CST6, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB019316; green = CST6, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CST6 IHC & IF
🧪Run skin first: cells in the corneal layer must stain (HPA: High in skin corneal-layer cells). Use adipose tissue as the negative tissue (HPA: adipocytes Not detected); cells outside the corneal layer on the skin slide should provide a background reference, although HPA does not assign them a staining level.
Positive control tissue: Skin (Cells in corneal layer, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CST6 in A-431, U-251MG, U2OS, with annotated localisation: Plasma membrane (approved) (HPA subcellular).
Technical controls: Run a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species and clonality, and a CST6 knockout control or cognate-peptide block if available (standard IHC practice). In skin, quench endogenous peroxidase and distinguish pigment from chromogen; block endogenous biotin if using biotin-based detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state a fixative (selected tissue-IHC caption). The caption uses Tris-EDTA at pH 9.0 for retrieval in tonsil, but does not establish whether skin staining depends on that retrieval condition (selected tissue-IHC caption). A matched frozen-section or IF protocol is not supplied, so whether either is easier than paraffin IHC is undetermined.
HPA tissue IHC evidence for CST6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data. Secreted protein, tissue location of RNA and protein is expected to differ.). 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 |
|---|---|---|---|---|
| Skin | Cells in corneal layer | High | Protein (IHC) | HPA → |
| Kidney | Collecting ducts | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CST6 IHC Tips
Use the documented paraffin section conditions as the IHC starting point, then judge CST6 staining against its secreted biology and tissue distribution (caption A07408-1; UniProt Q15828).
Which retrieval condition should I try first for CST6 in paraffin sections?
Start with heat-mediated retrieval in Tris-EDTA, pH 9.0 (datasheet A07408-1). This is the retrieval condition reported for the catalog antibody in paraffin-embedded human tonsil, where the primary antibody was applied at 1:200 overnight at 4°C (caption A07408-1). Keep section thickness, heating method, cooling period, and detection conditions consistent while comparing runs, since changing several variables obscures the cause of weak staining (standard IHC practice). If signal remains weak, compare a milder retrieval condition on adjacent sections and retain the condition that preserves morphology and yields a reproducible cellular pattern (standard IHC practice).
How should I handle fixation when CST6 staining varies between sections?
Target-specific fixation sensitivity is unknown: the catalog image describes paraffin-embedded human tonsil but does not state its fixative (caption A07408-1). Record each specimen's fixation method and duration, then compare sections processed with the same retrieval and 1:200 primary antibody condition before attributing differences to CST6 abundance (caption A07408-1; standard IHC practice). Examine morphology for poor preservation or excessive masking, and use identically processed control sections when troubleshooting a weak run (standard IHC practice). Do not infer a preferred fixative from skin staining or from CST6 secretion, glycosylation, or lack of a transmembrane segment (HPA tissue IHC; UniProt Q15828).
Where should convincing CST6 chromogenic staining appear?
Assess signal in the squamous epithelium of skin, where tissue IHC reports selective cytoplasmic expression and high staining in cells of the corneal layer (HPA tissue IHC). UniProt places CST6 in the secreted pathway and reports no transmembrane segment, so extracellular or surface-adjacent staining deserves evaluation alongside cellular staining (UniProt Q15828 topology). Kidney collecting ducts have medium reported staining, whereas adipocytes in adipose tissue are reported as undetected; these provide useful pattern comparisons when identically processed material is available (HPA tissue IHC). Compare the chromogen with a counterstained adjacent section to distinguish epithelial signal from pigment, folds, or detached surface material (standard IHC practice).
Could processing or glycosylation explain a changed CST6 staining pattern?
CST6 has a 1–28 signal peptide, a 29–149 mature chain, one annotated glycosylation site at residue 137, and no annotated isoforms (UniProt Q15828). Those features make epitope location relevant when judging whether a processed protein remains detectable, but the supplied catalog caption does not identify the antibody epitope (caption A07408-1). Ask for the mapped immunogen or epitope before assigning a staining difference to cleavage or glycosylation; neither mechanism is established by this IHC image (caption A07408-1; UniProt Q15828). Compare adjacent sections under identical retrieval and detection conditions, and treat a compartment change as an observation requiring independent confirmation (standard IHC practice).
How can I use IF to investigate an ambiguous CST6 IHC pattern?
Use IF as a companion localisation experiment, with a keratinocyte marker to identify the expected skin epithelial population (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and place the weaker CST6 signal in a far-red channel if tissue autofluorescence interferes, while checking single-stain and unstained controls (standard IF practice). Because CST6 is secreted and has no transmembrane segment, decide whether the question concerns accessible extracellular protein or an intracellular pool before adding permeabilisation; test mild permeabilisation only for the latter (UniProt Q15828 topology; standard IF practice). Interpret IF against the chromogenic section's cell pattern, and do not transfer its 1:200 paraffin-section dilution automatically to IF (caption A07408-1; standard IF practice).
What should I check when CST6 staining is diffuse or widespread?
First compare the suspect section with a no-primary control and an identically processed positive control to separate detection background from a failed run (standard IHC practice). In chromogenic IHC, check peroxidase blocking, secondary reagent specificity, wash stringency, and DAB development before increasing primary antibody concentration (standard IHC practice). The documented tonsil image used primary and secondary antibodies at 1:200, with the secondary applied for 30 minutes at room temperature; these conditions belong to that image (caption A07408-1). Because CST6 is secreted, diffuse extracellular color alone cannot establish cellular production; require a plausible tissue pattern and clean controls (UniProt Q15828; standard IHC practice).
How should I score CST6 across skin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored compartment before analysis: HPA reports selective cytoplasmic staining in skin squamous epithelium and high staining in cells of the corneal layer (HPA tissue IHC). For cellular signal, record the percentage of positive epithelial cells and an intensity-based H-score using the same thresholds across specimens (standard IHC practice). For extracellular or surface-associated chromogen, report positive area or optical density per mm² of evaluable epithelium separately, since CST6 is secreted (UniProt Q15828; standard IHC practice). Normalise to evaluable epithelial area or epithelial cell count, document excluded folds and damaged regions, and keep exposure and analysis settings fixed (standard IHC practice).
How do I distinguish true CST6 positivity from artefact?
A credible result follows the relevant anatomy: skin squamous epithelium shows selective cytoplasmic staining, while adipose tissue adipocytes are reported as undetected (HPA tissue IHC). CST6 is secreted and lacks a transmembrane segment, so surface-associated signal can be plausible, but isolated nuclear staining does not match its reported localisation (UniProt Q15828 topology; HPA tissue IHC). Discount color confined to section edges, folds, necrotic material, or regions also positive in a no-primary control; check endogenous peroxidase when using DAB (standard IHC practice). Interpret tonsil staining cautiously because the catalog caption documents its processing and antibody conditions, but supplies no cell-level localisation claim (caption A07408-1).
Boster reagents
Best CST6 / Cystatin-M IHC Antibodies
A07408-1 has a human tonsil paraffin-section IHC image (catalog image caption) and lists human IF reactivity (catalog applications/reactivity); no IF image is supplied (catalog IF images).
Real IHC data
Anti-Cystatin E/M CST6 Antibody
Cat # A07408-1
A07408-1 will render with its own human tonsil paraffin-section IHC figure (catalog image caption). It lists human reactivity and IF as an application, without an IF figure (catalog reactivity/applications/IF images).
Which to pick: For tissue IHC, choose A07408-1: its own paraffin-section image documents human tonsil, a 1:200 primary dilution, overnight incubation at 4°C, and Tris-EDTA pH 9 retrieval; the fixative is unreported (catalog image caption). For IF, A07408-1 lists a 1:50 dilution, but ICC is not listed and no IF figure is supplied (catalog applications/dilution/IF images). No cross-species choice is supported because A07408-1 lists human reactivity only; clonality is unreported (catalog reactivity/clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q15828 (CYTM_HUMAN, Cystatin-M).
- Human Protein Atlas. CST6 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CST6 subcellular location (ICC-IF): Mainly localized to the plasma membrane. In addition localized to the cytosol..
- Human Protein Atlas. CST6 antibody validation summary (2 antibodies).
- Predictive Values of Homeobox Gene A-Antisense Transcript 3 (HOXA-AS3), Cystatin 6 (CST6), and Chromobox Homolog 4 (CBX4) Expressions in Cancer Tissues for Recurrence of Early Colon Cancer After Surgery. International journal of general medicine 2024 — PMC10772051.
- Frequent loss of cystatin E/M expression implicated in the progression of prostate cancer. Oncogene 2009 — PMC2722685.
- Molecular clustering and prognostic features based on integrated databases predict survival and immune status in patients with gastric cancer. Frontiers in oncology 2025 — PMC12444767.
- PubMed PMID:8995380 — UniProt-cited evidence.
- PubMed PMID:9099741 — UniProt-cited evidence.
- PubMed PMID:12839564 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CST6 (Cystatin-M). Expected localisation Cytoplasmic in skin squamous epithelium (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A07408-1). Validated IHC antibody: A07408-1 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Skin (Cells in corneal layer); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A07408-1); 2. Fixation: Image fixative and duration unreported (datasheet A07408-1); 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 retrieval: Tris-EDTA pH 9.0 (datasheet A07408-1); 20 min, 95–100 °C (standard); 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-CST6, 1:100-1:300 (datasheet A07408-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Secreted CST6 may appear away from producing cells (UniProt; HPA tissue IHC); Isoform / epitope: No isoforms; mature chain 29–149 has no membrane-sided epitope (UniProt); Regulation: Psoriatic skin includes spinous-layer expression (UniProt). Evidence: UniProt Q15828, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the plasma membrane. In addition localized to…, Datasheet A07408-1, PMC10772051. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CST6/immunohistochemistry-ihc-guide.