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KRT16 / Keratin, type I cytoskeletal 16 · IHC design guide
Design Immunohistochemistry for KRT16
Plan KRT16 paraffin-section IHC around cytoplasmic staining in squamous epithelial cells, using tonsil as a medium-staining reference (HPA tissue IHC). Keep fixation consistent (standard IHC practice) and interpret results with HPA’s low antibody–RNA concordance in mind (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
KRT16 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 squamous epithelial cells (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in squamous epithelial cells (HPA tissue IHC) | |
| Antigen retrieval | No retrieval / mild HIER, citrate pH 6.0 if weak (rule: intermediate filament) | |
| Positive control | Tonsil | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M03393) | |
| Caveat | Low consistency between antibody staining and RNA expression (HPA tissue IHC) | |
| Regulation | Tissue-enhanced RNA expression (HPA tissue RNA) | |
| Isoform / epitope | No isoforms or processing annotated; epitope site unknown (UniProt) |
Section 1
Recommended KRT16 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published KRT16 protocols for tumor and skin sections (PMC8974228; PMC11900751; PMC13283874).
| Sample | Paraffin-embedded human skin tissue; fixative not specified (datasheet M03393) |
| Fixation | Image fixative and duration unreported (datasheet M03393); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | No retrieval needed for most intermediate-filament epitopes; if staining is weak, mild HIER in citrate buffer, pH 6.0, 10 min at 95 °C (standard rule: intermediate filament) |
| 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 monoclonal (clone EDC-11) anti-KRT16, 1:50 (datasheet M03393) |
| 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 | KRT16-positive staining in squamous epithelial cells of tonsil (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in squamous epithelial cells. No signal in the no-primary control. |
💡Decision noteStart without retrieval; if staining is weak, try mild citrate HIER at pH 6.0 (page retrieval rule: intermediate filament).
Section 2
What Is the Expected KRT16 Staining Pattern?
In paraffin IHC, expect KRT16 in the cytoplasm of squamous epithelial cells (HPA: tissue IHC profile), including medium staining in tonsillar squamous epithelium (HPA: Tonsil, Medium). KRT16 is a keratin intermediate filament protein with no transmembrane segment (UniProt P08779: function and topology). Interpret the pattern cautiously: HPA rates its tissue IHC profile Approved but reports low consistency between antibody staining and RNA expression (HPA: reliability description).
| Squamous epithelial cytoplasm stains in tonsil, with a moderate chromogenic signal. | This matches the reported positive cell population and level (HPA: Tonsil squamous epithelial cells, Medium). Score the epithelial compartment and its staining separately from neighboring cells; a tissue-wide score would obscure the cell-specific reference pattern (HPA: tissue IHC profile). |
| Strong nuclear or sharply membrane-restricted staining predominates in squamous cells. | That distribution conflicts with the reported cytoplasmic IHC profile (HPA: cytoplasmic expression in squamous epithelial cells) and the intermediate filament location seen by ICC-IF (HPA: subcellular summary). Check counterstain, detection deposits, and antibody controls before calling it KRT16 (standard IHC practice). |
| Adipocytes stain strongly, especially when the expected squamous pattern is absent. | Adipocytes were reported as Not detected in adipose tissue (HPA: Adipose tissue adipocytes, Not detected). Consider nonspecific binding or endogenous detection activity (standard IHC practice). Cell identity matters: HPA also reports Low staining in lung macrophages and skin fibroblasts (HPA: tissue IHC low entries). |
| Weak color spreads across epithelium, stroma, and empty areas without cell boundaries. | A widespread haze cannot establish the reported squamous cytoplasmic pattern (HPA: tissue IHC profile). Review the no-primary control, blocking, washes, and chromogen development for background sources (standard IHC practice); assess any remaining signal by cell type and compartment. |
| Tonsillar squamous epithelium has no detectable signal. | This misses a reported Medium positive reference (HPA: Tonsil squamous epithelial cells, Medium). Check section quality, retrieval, primary-antibody conditions, and detection with appropriate controls (standard IHC practice). One negative section does not resolve the profile's reported antibody–RNA inconsistency (HPA: reliability description). |
💡Expected KRT16 appearanceCall a positive result when squamous epithelial cytoplasm shows a discernible, approximately medium-intensity signal in tonsil (HPA: Tonsil, Medium; tissue IHC profile); predominant nuclear staining or strong adipocyte staining is discordant (HPA: cytoplasmic profile; Adipose adipocytes, Not detected).
| IHC reference population | Tonsillar squamous epithelial cells provide a Medium positive reference; oral mucosal squamous cells are reported Low (HPA: tissue IHC entries). Compare the same cell type, since a Low reference can be difficult to judge at a glance (standard IHC practice). |
| Interpretation of negative tissue | HPA reports Not detected in adipose-tissue adipocytes and bronchial respiratory epithelial cells (HPA: tissue IHC negative entries). Those calls concern the named cells; they do not certify every cell in either section as KRT16-negative. |
| Evidence strength | The tissue IHC profile is Approved, with low consistency between antibody staining and RNA expression (HPA: reliability and reliability description). Treat a matching image as support for the observed pattern, then use controls to investigate discordant samples (standard IHC practice). |
| Antibody validation by application | CAB000136 is listed as IHC Approved, while HPA000539 is ICC Approved with no IHC status supplied (HPA: antibody validation entries). Use the IHC validation when judging paraffin-section staining; the ICC entry does not establish IHC performance. |
| IF/ICC Q: Where should fluorescence appear? | A: HPA reports intermediate filament localization in ICC-IF, based on antibodies targeting proteins from multiple genes (HPA: subcellular summary and caution). This supports a filamentous cytoplasmic expectation, with that specificity caveat; it is not a paraffin IHC protocol. |
| Molecular context | KRT16 forms epithelial keratin intermediate filaments with type II keratins, including KRT6A or KRT6B (UniProt P08779: function and subunit). UniProt lists no transmembrane segment or signal peptide (UniProt P08779: topology and processing), consistent with an intracellular readout. |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in tonsillar squamous epithelium | A Medium signal is reported there (HPA: Tonsil); inadequate retrieval, primary incubation, or detection can produce a technical miss (standard IHC practice). | Run a known-positive section and detection controls alongside the sample; review retrieval and antibody conditions against the validated IHC procedure (standard IHC practice). |
| Nuclear signal dominates | Nuclear dominance conflicts with the cytoplasmic tissue profile (HPA: tissue IHC profile). Counterstain overlap or nonspecific detection can confuse interpretation (standard IHC practice). | Inspect the no-primary control and counterstain; score KRT16 only where signal follows the squamous cytoplasm (standard IHC practice; HPA: tissue IHC profile). |
| Strong staining in adipocytes | Adipocytes are Not detected in the HPA adipose-tissue reference (HPA: tissue IHC negative entry); nonspecific binding or endogenous detection activity is possible (standard IHC practice). | Confirm cell identity and compare a no-primary control; check blocking and the detection system before assigning target staining (standard IHC practice). |
| Diffuse brown haze obscures the epithelium | Background from insufficient washing or detection development can obscure a cell-specific pattern (standard IHC practice; HPA: squamous cytoplasmic profile). | Compare control and stained sections, review washes and chromogen timing, then reassess whether epithelial cytoplasm is distinguishable (standard IHC practice; HPA: tissue IHC profile). |
| Signal appears weaker than expected in oral mucosa | Oral mucosal squamous cells are reported Low, compared with Medium in tonsillar squamous cells (HPA: tissue IHC entries). | Use tonsillar squamous epithelium as the stronger reported reference; compare cell-specific staining under the same run conditions (HPA: tissue IHC entries; standard IHC practice). |
| ICC images and paraffin IHC seem different | ICC-IF resolves an intermediate filament location, while the tissue IHC report describes cytoplasmic staining (HPA: subcellular and tissue IHC summaries). | Judge each image against its own application and validation entry: CAB000136 is IHC Approved; HPA000539 is ICC Approved (HPA: antibody validation entries). |
Experiment evidence
IHC & IF images showing KRT16 staining
M03393 · IHCImmunohistochemical analysis of paraffin-embedded human skin, using Cytokeratin 16 Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000186832-KRT16/tissue/cerebral+cortex#imid_458234
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000186832-KRT16/tissue/colon#imid_458168
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000186832-KRT16/tissue/liver#imid_458161
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000186832-KRT16/tissue/kidney#imid_458162
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA000539; green = KRT16, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA000539; green = KRT16, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for KRT16 IHC & IF
🧪Run tonsil first: its squamous epithelial cells should stain for KRT16 (HPA: Medium in tonsil squamous epithelial cells). Run adipose tissue as the negative comparator (HPA: Not detected in adipocytes); on the tonsil slide, non-squamous cells should show background-level staining, but assess them as internal comparators rather than confirmed negatives (HPA: tonsil result specifies squamous epithelial cells).
Positive control tissue: Tonsil (Squamous epithelial cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show KRT16 in A-431, SiHa, hTCEpi, with annotated localisation: Intermediate filaments (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; an isotype control matched to the primary antibody’s host species and clonality; and a verified KRT16 knockout specimen or peptide-block control if the immunizing peptide is available (standard IHC practice). Block endogenous peroxidase before chromogenic detection in tonsil, where immune cells can contribute background (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and antigen retrieval dependence are unreported in the supplied evidence; the selected M03393 paraffin-section skin caption also does not state its fixative (caption: fixative not stated). The evidence does not establish whether frozen sections or IF would be easier; ICC-IF images exist for A-431, SiHa and hTCEpi, but the intermediate-filament localization summary carries a multiple-gene antibody caution (HPA: subcellular summary). For tonsil IHC, endogenous peroxidase in immune cells is a potential chromogenic artefact (standard IHC practice).
HPA tissue IHC evidence for KRT16
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 |
|---|---|---|---|---|
| Tonsil | Squamous epithelial cells | 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 KRT16 IHC Tips
Troubleshooting KRT16 staining in paraffin sections focuses on retrieval, cytoplasmic filament localisation, cell identity, and controls for chromogenic background.
What should I change if KRT16 staining is weak after antigen retrieval?
Start with no retrieval, then try mild HIER in citrate buffer at pH 6.0 if staining is weak (page rule: intermediate filament). Compare adjacent sections with and without retrieval, keeping antibody incubation and chromogenic development identical (standard IHC practice). Use paraffin-embedded human skin as a reference specimen because the catalog antibody has a tissue-IHC image in that preparation (catalog antibody M03393 caption). Check whether signal appears as cytoplasmic staining in squamous epithelium, the reported tissue pattern (HPA tissue IHC). If heating increases diffuse staining without improving that pattern, shorten retrieval and review background controls before increasing antibody concentration (standard IHC practice).
Could fixation explain weak or uneven KRT16 staining?
Target-specific sensitivity to fixation is unknown: the selected paraffin-section caption does not state a fixative (catalog antibody M03393 caption). Record the fixative, fixation duration, section thickness, and storage conditions for each block before comparing staining across specimens (standard IHC practice). On adjacent sections, compare the same retrieval and antibody conditions while changing only one processing variable at a time (standard IHC practice). A paraffin-embedded human skin image supports use of that preparation, but does not establish an optimal fixation time (catalog antibody M03393 caption). Judge any processing adjustment by preserved morphology and cytoplasmic squamous epithelial staining, rather than by overall stain intensity alone (HPA tissue IHC; standard IHC practice).
Where should convincing KRT16 staining appear in a tissue section?
Expect cytoplasmic staining in squamous epithelial cells in tissue sections (HPA tissue IHC). KRT16 contributes to epithelial keratin intermediate filament networks, so assess whether staining follows an intracellular filament-rich pattern rather than outlining cell membranes (UniProt P08779 function; HPA subcellular). HPA reports intermediate filament localisation from ICC/IF, with a caution that those antibodies target proteins from multiple genes (HPA subcellular). Compare epithelial staining with adjacent stromal and inflammatory cells on the same slide, using morphology and a counterstain to identify compartments (standard IHC practice). Treat isolated nuclear or extracellular deposits as suspect until controls and repeat sections support them (standard IHC practice).
How do I investigate an unexpected staining pattern or possible keratin cross-reactivity?
The supplied record lists 0 isoforms and an intermediate filament rod spanning residues 117–428 (UniProt P08779 record). It also lists no transmembrane segment or glycosylation sites, so a membrane-restricted pattern needs scrutiny (UniProt P08779 topology; UniProt P08779 glycosylation). Check the catalog antibody’s immunogen or mapped epitope, if available, against related keratins before attributing unexpected staining to KRT16 (standard IHC practice). KRT16 assembles with KRT6A or KRT6B, but their presence alone does not establish antibody specificity for KRT16 (UniProt P08779 subunit; standard IHC practice). Compare an independent antibody and appropriate tissue controls when specificity remains uncertain (standard IHC practice).
How can IF help check a puzzling chromogenic KRT16 result?
Use IF as a separate localisation check, pairing KRT16 with a marker that identifies the squamous epithelial cells being assessed (HPA tissue IHC; standard IF practice). Choose fluorophores after inspecting unstained tissue in each channel, and place the weaker signal in a channel with low tissue autofluorescence (standard IF practice). Because KRT16 is an intracellular filament protein with no transmembrane segment, permeabilise fixed cells or sections sufficiently for antibody access (UniProt P08779 function; UniProt P08779 topology; standard IF practice). Include single-label and secondary-only controls to assess bleed-through and nonspecific fluorescence (standard IF practice). Compare the IF compartment pattern with the chromogenic section; HPA reports intermediate filaments but cautions about antibody specificity (HPA subcellular).
How do I reduce diffuse or patchy chromogenic background?
First compare a no-primary control with the test section to locate signal arising from the detection system (standard IHC practice). For peroxidase-based detection, include a peroxide block of about 10–20 minutes and rinse thoroughly before primary antibody incubation (standard IHC practice). Use an appropriate protein block, wash between steps, and shorten DAB development if background rises before epithelial detail becomes clear (standard IHC practice). Inspect section edges, folds, and damaged areas separately from intact epithelium because reagent trapping can mimic staining (standard IHC practice). Accept a change only when cytoplasmic squamous epithelial signal remains distinguishable from the control (HPA tissue IHC; standard IHC practice).
What is a defensible way to quantify KRT16-positive cells? ⚠ ANSWER MARKED FOR VERIFICATION
Define the squamous epithelial compartment before scoring, since that is the reported site of cytoplasmic KRT16 tissue staining (HPA tissue IHC). For comparable sections, report the percentage of positive epithelial cells and, if intensity is reproducible, an H-score from 0–300 (standard IHC practice). Count positive cells per mm² only when the measured epithelial area is recorded, and normalise counts to that area (standard IHC practice). Keep illumination, threshold, counterstain handling, and DAB development consistent across slides (standard IHC practice). Exclude folds, necrosis, and section edges using preset criteria, then document the excluded area and scoring rules (standard IHC practice).
When should a positive-looking KRT16 stain be questioned?
A convincing result places staining in the cytoplasm of morphologically identified squamous epithelial cells (HPA tissue IHC). Question predominantly nuclear, membrane-only, or extracellular staining because KRT16 is an intracellular intermediate filament component without a transmembrane segment (UniProt P08779 function; UniProt P08779 topology). Check whether staining is confined to edges, folds, or necrotic areas, where section artefacts can dominate (standard IHC practice). For peroxidase detection, compare a no-primary control to identify endogenous enzyme activity or detection-related deposits (standard IHC practice). Interpret weak signals cautiously because HPA rates its tissue staining Approved but reports low consistency with RNA expression data (HPA tissue IHC).
Boster reagents
Best KRT16 / Keratin, type I cytoskeletal 16 IHC Antibodies
One human-reactive anti-KRT16 antibody has an IHC image from paraffin-embedded human skin (catalog reactivity: Human; catalog IHC image caption). IF/ICC is listed without an IF image (catalog applications; catalog IF image alts: none).
Real IHC data
Anti-Cytokeratin 16 KRT16 Rabbit Monoclonal Antibody
Cat # M03393
M03393 is listed for human IHC, IF and ICC (catalog applications; catalog reactivity: Human). Its IHC image shows paraffin-embedded human skin; no IF image is supplied (catalog IHC image caption; catalog IF image alts: none).
Which to pick: For human paraffin-section IHC, choose M03393: it is a rabbit monoclonal with an IHC image from paraffin-embedded human skin (catalog host: Rabbit; catalog clone: EDC-11; catalog IHC image caption). The caption does not report the fixative (catalog IHC image caption). M03393 is also listed for human IF/ICC, but no IF image is supplied; its Human-only reactivity provides no basis for a cross-species choice (catalog applications; catalog IF image alts: none; catalog reactivity: Human).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P08779 (K1C16_HUMAN, Keratin, type I cytoskeletal 16).
- Human Protein Atlas. KRT16 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. KRT16 subcellular location (ICC-IF): Localized to the intermediate filaments. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. KRT16 antibody validation summary (2 antibodies).
- BarH-like homeobox 2 represses the transcription of keratin 16 and affects Ras signaling pathway to suppress nasopharyngeal carcinoma progression. Bioengineered 2022 — PMC8974228.
- Fibroblast Growth Factor Receptor 2 (FGFR2) Is Required for Meibomian Gland Homeostasis in the Adult Mouse. Investigative ophthalmology & visual science 2017 — PMC5444547.
- Silencing KRT16 inhibits keratinocyte proliferation and VEGF secretion in psoriasis via inhibition of ERK signaling pathway. The Kaohsiung journal of medical sciences 2019 — PMC11900751.
- Differentiation defects reposition sebaceous glands as inflammatory instigators in the early pathogenesis of hidradenitis suppurativa. Frontiers in immunology 2026 — PMC13283874.
- PubMed PMID:2431270 — UniProt-cited evidence.
- PubMed PMID:2451124 — UniProt-cited evidence.
- PubMed PMID:7487986 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for KRT16 (Keratin, type I cytoskeletal 16). Expected localisation Cytoplasmic in squamous epithelial cells (HPA tissue IHC). Antigen retrieval: No retrieval / mild HIER, citrate pH 6.0 if weak (rule: intermediate filament). Validated IHC antibody: M03393 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Tonsil (Squamous epithelial cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human skin tissue; fixative not specified (datasheet M03393); 2. Fixation: Image fixative and duration unreported (datasheet M03393); 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: No retrieval needed for most intermediate-filament epitopes; if staining is weak, mild HIER in citrate…; 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 monoclonal (clone EDC-11) anti-KRT16, 1:50 (datasheet M03393); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M03393); Caveat: Low consistency between antibody staining and RNA expression (HPA tissue IHC); Isoform / epitope: No isoforms or processing annotated; epitope site unknown (UniProt); Regulation: Tissue-enhanced RNA expression (HPA tissue RNA). Evidence: UniProt P08779, HPA tissue IHC · Approved, HPA ICC-IF · intermediate filaments. Caution: Based on antibodies targeting…, Datasheet M03393, PMC8974228, PMC11900751. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/KRT16/immunohistochemistry-ihc-guide.