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KLK4 / Kallikrein-4 · IHC design guide
Design Immunohistochemistry for KLK4
Plan KLK4 IHC-P using high, granular staining in prostate glandular cells as a positive reference (HPA tissue IHC). Interpret staining in light of KLK4 secretion and precursor cleavage (UniProt), and keep fixation consistent across sections (standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
KLK4 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 | Secreted (UniProt); IHC compartment unassigned (HPA tissue IHC) | |
| Staining pattern | Granular staining in prostate glandular cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Prostate | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Secretion may separate staining from producer cells (UniProt; HPA tissue IHC) | |
| Regulation | Prostate-enriched expression (HPA tissue IHC) | |
| Isoform / epitope | 2 isoforms; aa 1–30 are cleaved during maturation (UniProt) |
Section 1
Recommended KLK4 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with a published mouse mandibular incisor IHC protocol (PMC7255463 methods).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A03430); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-KLK4, 1:100–1:500 starting range (standard) |
| 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 | KLK4-positive staining in glandular cells of prostate (HPA tissue IHC: High). HPA tissue profile: Granular expression in prostate. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen retrieval; PMC7255463 methods).
Section 2
What Is the Expected KLK4 Staining Pattern?
KLK4 is secreted and has no transmembrane segment (UniProt Q9Y5K2 topology). In paraffin-section IHC, expect predominantly granular staining associated with prostate glandular cells, where HPA reports high expression (HPA: prostate glandular cells, High; granular expression). HPA rates the tissue staining Enhanced, with high consistency between antibody staining and RNA expression, while cautioning that secretion can separate protein and RNA locations (HPA: Enhanced reliability description).
| Granular chromogenic signal in prostate glandular cells, strongest where the glandular epithelium is identifiable (HPA: granular prostate expression; glandular cells, High). | This matches the reported tissue and cell pattern (HPA: prostate glandular cells, High). Score the glandular-cell pattern and intensity; secretion means adjacent material may contain KLK4, but HPA does not define a required luminal staining pattern (UniProt Q9Y5K2: secreted; HPA: reliability description). |
| A sharply nuclear or continuous plasma-membrane pattern dominates, with little granular glandular-cell staining (HPA: granular prostate expression). | Treat this as a localization mismatch requiring validation, rather than a characteristic KLK4 pattern: UniProt describes KLK4 as secreted with no transmembrane segment (UniProt Q9Y5K2 topology). Compare with a positive prostate section and a detection-only control (general IHC practice). |
| Strong staining centers on adipocytes, hematopoietic cells, or other cells outside the reported prostate glandular-cell pattern (HPA: prostate glandular cells, High). | Assess possible antibody cross-reactivity or endogenous detection activity (general IHC practice). HPA reports KLK4 as not detected in adipose adipocytes and bone-marrow hematopoietic cells; those observations are cell- and tissue-specific, not proof that every other cell must be negative (HPA: tissue IHC). |
| Diffuse color covers tissue, stroma, and slide regions without a discernible granular glandular-cell pattern (HPA: granular prostate expression). | This is hard to score as specific KLK4 staining (HPA: granular prostate expression). Check blocking, washes, chromogen development, and a detection-only control as general IHC troubleshooting; extracellular signal alone is inconclusive because KLK4 is secreted (UniProt Q9Y5K2: secreted). |
| No discernible staining appears in an adequately preserved prostate glandular-cell positive control (HPA: prostate glandular cells, High). | A negative run is not persuasive until the positive control works (general IHC practice). Check tissue identity, retrieval conditions, antibody and detection steps, and section quality; the supplied sources report no KLK4-specific fixation sensitivity or required retrieval condition (HPA: tissue IHC; UniProt Q9Y5K2). |
💡Expected KLK4 appearanceCall a result consistent with KLK4 when high, predominantly granular staining is associated with prostate glandular cells (HPA: prostate glandular cells, High; granular expression); dominant nuclear, continuous membrane, or indiscriminate diffuse color is a suspected false-positive pattern requiring controls (UniProt Q9Y5K2 topology; general IHC practice).
| Tissue and cell context (HPA: tissue IHC) | Prostate glandular cells are the reported high-staining reference; listed negative cells include adipocytes and bone-marrow hematopoietic cells (HPA: tissue IHC). Compare like cell types when scoring. |
| Secretion and topology (UniProt Q9Y5K2) | KLK4 is secreted and lacks a transmembrane segment (UniProt Q9Y5K2 topology). Protein may be spatially separated from expressing cells; HPA explicitly cautions that RNA–protein location correlation is complex for secreted variants (HPA: reliability description). |
| Precursor processing (UniProt Q9Y5K2) | The annotated precursor has a signal peptide at residues 1–26, propeptide at 27–30, and KLK4 chain at 31–254 (UniProt Q9Y5K2 processing). Epitope location is unspecified, so these annotations cannot predict which processed forms this antibody detects. |
| Isoforms and glycosylation (UniProt Q9Y5K2) | UniProt lists 2 isoforms and one glycosylation site at residue 169 (UniProt Q9Y5K2). The supplied evidence does not map the antibody epitope or establish isoform-specific staining or an IHC effect of glycosylation. |
| Validation boundary (HPA: Enhanced tissue reliability; HPA051839 IHC Enhanced) | HPA reports consistency between tissue staining and RNA data and lists HPA051839 as IHC Enhanced (HPA: tissue IHC; antibody validation). That supports the reported prostate pattern; it does not specify a retrieval recipe, fixation effect, or ICC-IF image pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Prostate positive control is blank or unexpectedly weak (HPA: prostate glandular cells, High). | Possible section, retrieval, antibody-incubation, or detection failure (general IHC practice); the sources give no KLK4-specific fixation or retrieval response. | Confirm glandular tissue on the counterstain, then inspect control performance and each IHC step (general IHC practice). Do not interpret experimental negatives until the positive control stains. |
| Color is widespread across cells and stroma rather than granular in glandular cells (HPA: granular prostate expression). | Background from detection reagents, insufficient blocking or washing, or excessive chromogen development is possible (general IHC practice); secretion alone does not establish that all diffuse color is specific (UniProt Q9Y5K2: secreted). | Compare detection-only and positive controls; adjust blocking, washes, or chromogen timing according to the assay (general IHC practice). Reassess whether a cell-associated pattern remains. |
| Unexpected cells stain strongly, including cells in an HPA-listed negative tissue (HPA: tissue IHC). | Cross-reactivity or endogenous detection activity is possible (general IHC practice). For HRP-based detection, endogenous peroxidase is one possible contributor (general IHC practice). | Review a detection-only control and the positive prostate control; check cell identity on the counterstain (general IHC practice). Treat HPA negative calls as comparisons for the listed cells, not universal tissue exclusions (HPA: tissue IHC). |
| A nuclear or continuous membrane outline dominates the slide (UniProt Q9Y5K2 topology). | This conflicts with the reported secreted, non-transmembrane protein and granular prostate tissue pattern (UniProt Q9Y5K2 topology; HPA: granular prostate expression); nonspecific signal is possible. | Check a detection-only control, compare against granular glandular-cell staining in prostate, and record the compartment separately from overall intensity (general IHC practice; HPA: tissue IHC). |
| A granular prostate signal is accompanied by apparent luminal or extracellular color (HPA: granular prostate expression). | A secreted protein can appear beyond its producing cells (UniProt Q9Y5K2: secreted; HPA: reliability description), but the supplied HPA record does not establish a required luminal pattern. | Score the documented glandular-cell pattern first (HPA: prostate glandular cells, High). Use controls and tissue morphology to assess the additional color, and report its location without calling it KLK4-specific solely from secretion. |
| Can this IHC pattern be used as an ICC-IF acceptance criterion? (HPA: subcellular ICC-IF record) | HPA summarizes KLK4 as secreted but provides no main subcellular location or cell-line ICC-IF images (HPA: subcellular ICC-IF record). | Use this page's prostate pattern for IHC interpretation (HPA: tissue IHC). Establish ICC-IF localization and controls on its separate guide page; the supplied record does not support an ICC-IF protocol or definitive intracellular compartment. |
Experiment evidence
IHC & IF images showing KLK4 staining
A03430 · IHCImmunohistochemistry of KLK4 in human kidney tissue with KLK4 antibody at 5 μg/mL.
A03430 · IFImmunofluorescence of KLK4 in human kidney tissue with KLK4 antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000167749-KLK4/tissue/cerebral+cortex#imid_14434881
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000167749-KLK4/tissue/colon#imid_14430482
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000167749-KLK4/tissue/liver#imid_14430483
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000167749-KLK4/tissue/kidney#imid_14430476
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for KLK4 IHC & IF
🧪Run prostate first: glandular cells should stain (HPA: High in prostate glandular cells). Use adrenal gland as a negative tissue (HPA: Not detected in adrenal gland glandular cells); on the prostate slide, nonglandular cells should remain near background and provide an internal comparison.
Positive control tissue: Prostate (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for KLK4; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) slide, an isotype control matched to the primary antibody’s host species and clonality, and, if available, a KLK4 knockout specimen or immunizing-peptide block. For chromogenic IHC, quench endogenous peroxidase and check the no-primary slide for residual signal (standard IHC practice).
⚠️Feasibility: A KLK4-specific fixation window or fixation effect is unreported, and the selected A03430 kidney IHC caption does not state a fixative (caption: fixative unreported). Retrieval dependence is unreported; the supplied evidence does not establish whether frozen sections or IF are easier, and HPA lists no ICC-IF image cell lines (HPA subcellular record). No prostate-specific artefact is documented; assess luminal staining separately from glandular-cell staining because KLK4 is secreted (UniProt Q9Y5K2: Secreted).
HPA tissue IHC evidence for KLK4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data. At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex.). 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 |
|---|---|---|---|---|
| Prostate | Glandular cells | High | 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 KLK4 IHC Tips
Troubleshoot chromogenic KLK4 IHC in paraffin sections by checking retrieval, staining distribution, controls, and scoring before interpreting signal.
How should I adjust retrieval when KLK4 staining is weak or uneven?
Start with citrate pH 6.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page IHC retrieval). Run a prostate control alongside test sections, because prostate glandular cells show high KLK4 staining (HPA tissue IHC), and keep section thickness, cooling, and detection conditions consistent between runs (standard IHC practice). If staining remains weak, vary retrieval time in a controlled comparison before trying another buffer or pH (standard IHC practice). Check whether stronger retrieval raises staining in the expected glandular cells without also raising diffuse background or damaging tissue architecture (HPA tissue IHC; standard IHC practice).
Could fixation explain inconsistent KLK4 staining across paraffin sections?
KLK4-specific sensitivity to fixation is unknown from the supplied evidence; the selected kidney image caption does not state a fixative (selected A03430 tissue-IHC caption). Record each specimen’s fixative, fixation duration, processing history, and section age, then compare sections processed together with the same retrieval and detection conditions (standard IHC practice). Include prostate glandular tissue as a positive reference because its staining is high in the HPA tissue profile (HPA tissue IHC). If staining differs between processing batches, repeat the comparison under matched conditions before attributing the difference to KLK4 expression (standard IHC practice). Neither the tissue profile nor the protein’s secretion and glycosylation annotations establish a fixation effect (HPA tissue IHC; UniProt Q9Y5K2).
What staining pattern should I expect for KLK4 in tissue?
Interpret KLK4 as a secreted protein with no annotated transmembrane segment, so staining may occur outside producing cells as well as within them (UniProt Q9Y5K2 topology). In prostate, look for glandular cell staining and assess whether its distribution resembles the reported granular expression profile (HPA tissue IHC). Evaluate luminal or extracellular signal in relation to adjacent glandular cells and tissue structure rather than counting it automatically as positive cells (UniProt Q9Y5K2 subcellular location; standard IHC practice). Compare serial sections and a primary-antibody omission control when a sharply nuclear or uniformly membrane-bound pattern dominates, because those patterns need additional specificity checks for a secreted target (UniProt Q9Y5K2 topology; standard IHC practice).
Could KLK4 processing or isoforms change what my antibody detects?
Map the antibody’s stated epitope, if available, against the 254-aa precursor, signal peptide at residues 1–26, propeptide at 27–30, and mature chain at 31–254 (UniProt Q9Y5K2 processing). KLK4 has 2 annotated isoforms and a glycosylation site at residue 169, so document which sequence region the reagent is intended to recognise (UniProt Q9Y5K2 isoforms and glycosylation). An epitope within a removed segment may produce a different tissue pattern from one retained in the mature chain (UniProt Q9Y5K2 processing; standard immunostaining principle). If epitope information is unavailable, compare staining with a separately validated epitope or orthogonal expression evidence before assigning a pattern to one isoform (standard IHC practice).
How can I check an ambiguous KLK4 IHC pattern by immunofluorescence?
Use IF as a separate validation experiment for an ambiguous chromogenic pattern, while keeping the IHC result anchored to its own controls (standard IHC/IF practice). In prostate, multiplex KLK4 with a validated glandular-cell marker to test whether signal lies in the expected cell population (HPA tissue IHC; standard IF practice). Choose fluorophores after inspecting unstained tissue autofluorescence, favouring channels with clear separation from that background, and include single-label controls (standard IF practice). Set permeabilisation according to the mapped epitope and whether fixation leaves it accessible: intracellular epitopes may require it, whereas accessible extracellular epitopes may not (UniProt Q9Y5K2 topology; standard IF practice). No KLK4 ICC/IF cell-line images are listed in the supplied HPA record (HPA subcellular record).
How do I distinguish KLK4 signal from chromogenic background?
Check a section with the primary antibody omitted to reveal detection-system and tissue background, and assess any staining at folds, edges, or damaged areas separately (standard IHC practice). Apply a peroxidase block before peroxidase-based chromogenic detection and inspect whether residual endogenous activity could account for the deposit (standard IHC practice). Titrate the primary antibody and detection exposure together rather than interpreting a darker stain alone as stronger KLK4 expression (standard IHC practice). Use prostate glandular cells as a positive reference and adipose-tissue adipocytes as a reported undetected comparison, while allowing for differences in specimen handling (HPA tissue IHC; standard IHC practice). The selected kidney caption reports 5 µg/mL for A03430, but gives no fixation condition (selected A03430 tissue-IHC caption).
How should I score KLK4 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue compartment and eligible cells before scoring, because KLK4 is secreted and extracellular staining can confound a cell-based measure (UniProt Q9Y5K2 subcellular location; standard IHC practice). For prostate glandular cells, record the percentage positive and intensity categories, then calculate an H-score consistently across cases if that is the study’s chosen endpoint (HPA tissue IHC; standard IHC practice). For predominantly extracellular deposits, measure positive area or signal density per mm² within a predefined compartment instead of assigning that signal to individual cells (standard IHC practice). Normalise cell-based scores to eligible glandular cells and area-based scores to evaluable tissue area, excluding folds and necrosis by a prespecified rule (standard IHC practice). Keep retrieval, staining batch, imaging, and thresholds consistent (standard IHC practice).
When is an unexpected KLK4-positive region credible?
Give greater weight to a reproducible pattern in prostate glandular cells, where KLK4 staining is reported as high, while recognising that a secreted protein can appear away from its producing cells (HPA tissue IHC; UniProt Q9Y5K2 subcellular location). Treat staining confined to an unexpected cell type or exclusively nuclear compartment as a finding that requires validation, given KLK4’s secreted annotation and lack of a transmembrane segment (UniProt Q9Y5K2 topology; standard IHC practice). Recheck edge effects, necrotic areas, and residual endogenous peroxidase activity with matched morphology and detection controls (standard IHC practice). The selected A03430 caption shows human kidney staining at 5 µg/mL, but does not establish the fixative or make every kidney signal specific (selected A03430 tissue-IHC caption; standard IHC practice).
Boster reagents
Best KLK4 / Kallikrein-4 IHC Antibodies
A03430 has human IHC-P and IF validation (catalog: applications and reactivity), with IHC and IF images from human kidney tissue (catalog: image captions).
Real IHC data
Anti-Kallikrein-4 KLK4 Antibody
Cat # A03430
A03430 is listed for human IHC-P and IF (catalog: applications and reactivity). Its images show human kidney tissue stained by IHC at 5 μg/mL and IF at 20 μg/mL (catalog: IHC and IF image captions).
Which to pick: For tissue IHC, choose A03430: it is listed for IHC-P, and its own IHC image shows human kidney tissue; the fixative is unreported (catalog: applications and IHC image caption). For IF, A03430 has human kidney tissue image data, but ICC validation is unreported (catalog: applications and IF image caption). Cross-species use is unvalidated because the listed reactivity is human only; the host is rabbit, and clonality is unreported (catalog: reactivity, host and clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y5K2 (KLK4_HUMAN, Kallikrein-4).
- Human Protein Atlas. KLK4 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. KLK4 subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. KLK4 antibody validation summary (1 antibodies).
- Clinical significance of kallikrein-related peptidase-4 in oral cancer. Anticancer research 2015 — PMC4577232.
- A single cell transcriptional profile of benign prostatic hyperplasia. Scientific reports 2026 — PMC13009279.
- Morphological, Histochemical, and Proteomic Analysis of the Effects of Fluoride and Amoxicillin, with Calcium and Vitamin D Supplementation, on Dental Enamel Formation. Calcified tissue international 2026 — PMC13201332.
- Effects of applying amoxicillin in juvenile mice on enamel mineralization and the expression of kallikrein‑related peptidase 4 and tight junction proteins in ameloblasts. International journal of molecular medicine 2020 — PMC7255463.
- PubMed PMID:10077646 — UniProt-cited evidence.
- PubMed PMID:10485467 — UniProt-cited evidence.
- PubMed PMID:10438493 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for KLK4 (Kallikrein-4). Expected localisation Secreted (UniProt); IHC compartment unassigned (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A03430 at 1:100–1:500 (standard starting range) (the product's own IHC validation image is shown). Controls (HPA): positive Prostate (Glandular cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A03430); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-KLK4, 1:100–1:500 starting range (standard); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Secretion may separate staining from producer cells (UniProt; HPA tissue IHC); Isoform / epitope: 2 isoforms; aa 1–30 are cleaved during maturation (UniProt); Regulation: Prostate-enriched expression (HPA tissue IHC). Evidence: UniProt Q9Y5K2, HPA tissue IHC · Enhanced, HPA ICC-IF · Secreted, Datasheet A03430, PMC7255463. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/KLK4/immunohistochemistry-ihc-guide.