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KLK4 / Kallikrein-4 · IHC design guide
Design Immunohistochemistry for KLK4
Plan KLK4 chromogenic IHC in paraffin sections using granular staining in prostate glandular cells as a reference (HPA tissue IHC). This guide covers fixation, retrieval, controls and scoring while accounting for KLK4 secretion (UniProt).
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; tissue staining compartment unspecified (UniProt; 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 formalin fixation consistent across paraffin sections (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 A03430) | |
| Caveat | Secreted KLK4 may stain beyond producing cells (UniProt; HPA tissue IHC) | |
| Regulation | Prostate-enriched RNA expression (HPA tissue RNA) | |
| Isoform / epitope | 2 isoforms; cleaved regions affect epitope choice (UniProt) |
Section 1
Recommended KLK4 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by three published KLK4 IHC methods (PMC13009279; PMC13201332; PMC7255463).
| 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 (this page’s antigen retrieval setting).
Section 2
What Is the Expected KLK4 Staining Pattern?
KLK4 is a secreted protein with no transmembrane segment (UniProt Q9Y5K2 topology). In paraffin sections, look for granular staining in prostate glandular cells, where staining is reported as High (HPA: prostate tissue IHC). HPA rates the tissue pattern Enhanced, citing consistency between antibody staining and RNA expression; secretion means the protein signal need not coincide exactly with the cells expressing its RNA (HPA: tissue reliability).
| Granular signal in prostate glandular cells. | This matches the reported positive pattern and High staining level (HPA: prostate tissue IHC). Judge it against the tissue architecture and negative controls; the secreted annotation alone does not specify every stained subcellular compartment (UniProt Q9Y5K2; HPA: subcellular summary). |
| Predominantly nuclear staining in prostate glandular cells. | A nuclear dominant pattern does not match the reported granular tissue pattern or secreted annotation (HPA: prostate tissue IHC; UniProt Q9Y5K2 topology). Treat it as suspect until the assay controls and an independently validated staining pattern support it. |
| Strong staining in cells reported as negative, such as adrenal glandular cells. | HPA reports KLK4 as Not detected in those cells (HPA: adrenal gland tissue IHC). Unexpected signal may reflect antibody cross-reactivity or endogenous chromogenic detection activity; assess the staining with appropriate controls before assigning it to KLK4. |
| Uniform haze across cells and surrounding tissue. | This lacks the reported granular, glandular pattern (HPA: prostate tissue IHC). Diffuse background can arise from nonspecific binding, incomplete blocking, or detection reagent background (general IHC practice). Its presence limits interpretation of a weak apparent positive signal. |
| No staining in prostate glandular cells. | This conflicts with the reported High positive tissue result (HPA: prostate tissue IHC). Check control performance and the actual antibody's IHC-P conditions before treating the section as biologically negative; HPA supplies no KLK4 specific fixation sensitivity here. |
💡Expected KLK4 appearanceCall a section positive when prostate glandular cells show clear granular staining at the reported High level (HPA: prostate tissue IHC); nuclear dominant signal or comparable staining in HPA negative cells warrants control based review (UniProt Q9Y5K2 topology; HPA: negative tissue IHC).
| Secreted protein and processing | KLK4 has a signal peptide at residues 1–26, a propeptide at 27–30, and a mature chain at 31–254 (UniProt Q9Y5K2). Interpret staining with secretion in mind; these annotations do not establish where a particular antibody's epitope lies. |
| Prostate reference tissue | HPA reports granular expression and High glandular cell staining in prostate (HPA: prostate tissue IHC). Use that observed pattern as the positive benchmark; HPA notes that secreted protein location and RNA location may differ (HPA: tissue reliability). |
| Negative tissue comparison | HPA reports Not detected in adrenal glandular cells and appendix glandular cells, among other listed examples (HPA: negative tissue IHC). These are comparison patterns, not proof that every cell in every specimen must remain unstained. |
| Strength and limits of validation | The tissue profile is rated Enhanced and HPA051839 has Enhanced IHC validation (HPA: tissue reliability; HPA: antibody validation). Neither entry supplies antigen retrieval settings, an epitope map, or a KLK4 specific fixation effect. |
| IF/ICC: what pattern is established? | HPA summarizes KLK4 as secreted but provides no main subcellular location, ICC-IF images, or ICC validation for HPA051839 (HPA: subcellular summary; HPA: antibody validation). The IHC glandular pattern cannot by itself establish an IF/ICC staining pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Prostate positive control is blank. | A failed IHC step or unsuitable antibody conditions are possibilities (general IHC practice); the result conflicts with High prostate glandular staining (HPA: prostate tissue IHC). | Confirm section integrity and detection controls, then check the actual IHC-validated antibody's recommended retrieval, dilution, and detection conditions. No KLK4 specific retrieval or dilution is supplied here. |
| Prostate signal is weak or hard to distinguish from background. | Poor signal separation can obscure the reported granular glandular pattern (HPA: prostate tissue IHC; general IHC practice). HPA does not establish a KLK4 specific fixation cause. | Compare with a clean negative control and review the antibody's IHC-P conditions. Adjust general assay variables one at a time, documenting whether glandular granules become distinguishable. |
| The slide shows mainly nuclear staining. | The compartment is inconsistent with KLK4's secreted annotation and HPA's granular prostate pattern (UniProt Q9Y5K2 topology; HPA: prostate tissue IHC). | Inspect matched controls and antibody specificity evidence. Do not score nuclear dominant signal as the expected KLK4 pattern solely because it appears in prostate tissue. |
| Adrenal or appendix glandular cells stain strongly. | Both are listed as Not detected (HPA: negative tissue IHC). Cross-reactivity or endogenous chromogenic activity may contribute (general IHC practice). | Review a no primary antibody control and the relevant endogenous activity block; compare staining distribution with the prostate positive control before interpreting unexpected signal. |
| Color develops broadly outside glandular cells. | Widespread color may reflect detection background or nonspecific binding (general IHC practice), rather than the granular prostate glandular pattern (HPA: prostate tissue IHC). | Check no primary and reagent controls, blocking and wash steps, and chromogen development. Repeat scoring only where cell boundaries and granular signal remain interpretable. |
| An IF/ICC result is used to justify the IHC pattern. | HPA lists no ICC-IF images or ICC validation for the listed antibody (HPA: subcellular summary; HPA: antibody validation). | Keep the IHC interpretation anchored to the HPA prostate tissue pattern. Evaluate any IF/ICC experiment under its separate guide and controls; do not infer an IF protocol from the IHC evidence. |
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 the negative tissue (HPA: Not detected in adrenal gland glandular cells), and assess nonglandular cells on the prostate slide for background rather than assuming they are KLK4-negative.
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 control and an isotype control matched to the primary antibody’s host and immunoglobulin class; use KLK4 knockout material or a validated immunogen-peptide block as a biological specificity control. For chromogenic prostate IHC, quench endogenous peroxidase and assess endogenous biotin if using avidin–biotin detection.
⚠️Feasibility: The supplied evidence reports no target-specific fixation window or retrieval dependence, so optimize antigen retrieval empirically; the fixative is unreported in the selected A03430 kidney IHC caption (A03430 caption). These data do not establish whether frozen sections or IF/ICC are easier, and HPA lists no ICC-IF cell-line images (HPA subcellular). Because KLK4 is secreted (UniProt Q9Y5K2 subcellular), luminal or extracellular signal in prostate may complicate assignment to individual glandular cells (HPA: High in prostate glandular cells).
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 KLK4 staining in paraffin sections by checking retrieval, controls, cellular distribution and scoring before interpreting chromogenic signal.
What retrieval should I try first when KLK4 staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval rule). Let sections cool in buffer before washing, and compare them with a known positive prostate section processed in the same run (HPA: High in prostate glandular cells; standard IHC practice). If staining remains weak, test a second retrieval condition on matched sections while holding antibody concentration and detection constant (standard IHC practice). Score signal and tissue damage together: stronger staining is useful only if glandular detail remains intact and the no-primary control stays clear (standard IHC practice).
Could fixation explain weak KLK4 staining in my paraffin sections?
KLK4-specific fixation sensitivity is unknown because no target-specific fixation conditions or comparison are supplied (evidence payload: fixation not stated). Record the fixative and fixation interval for each block, then compare blocks using the same pH 6.0 citrate retrieval and detection run (page retrieval rule; standard IHC practice). Poor morphology or uneven staining across a section can indicate a processing problem, but neither pattern establishes a KLK4-specific fixation effect (standard IHC practice). Include a prostate glandular positive control so that a weak study section can be assessed against staining obtained in that run (HPA: High in prostate glandular cells; standard IHC practice).
Where should convincing KLK4 staining appear in chromogenic IHC?
KLK4 is annotated as secreted, with a signal peptide at residues 1–26 and no transmembrane segment (UniProt Q9Y5K2 topology). In prostate, assess staining in glandular cells and note whether signal also extends into adjacent extracellular or luminal material; a continuous membrane rim is not required by its topology (HPA: High in prostate glandular cells; UniProt Q9Y5K2 topology). Compare each compartment with a no-primary control and the tissue morphology before calling extracellular deposits positive (standard IHC practice). The selected kidney IHC image shows staining at 5 µg/mL, but its caption does not establish a normal localisation pattern or state a fixative (A03430 image caption).
Can processing or isoforms change what the antibody detects in sections?
KLK4 has 2 annotated isoforms, while the supplied evidence does not map this antibody's epitope to either one (UniProt Q9Y5K2 isoforms; evidence payload: epitope unspecified). Its precursor contains a signal peptide at residues 1–26, a propeptide at 27–30, and a processed chain at 31–254 (UniProt Q9Y5K2 processing). A glycosylation site is annotated at residue 169, so document the antibody's mapped immunogen or epitope before attributing a staining difference to processing or glycosylation (UniProt Q9Y5K2 glycosylation; standard IHC practice). Until that mapping is available, report the observed staining compartment and control results without assigning it to a particular isoform or processed form (standard IHC practice).
How should I check KLK4 localisation in a companion IF experiment?
For companion IF, multiplex KLK4 with a glandular epithelial marker to identify the expected prostate cell population (HPA: High in prostate glandular cells; standard IF practice). Choose a red or far-red fluorophore after imaging an unstained section, since tissue autofluorescence can obscure weaker channels (standard IF practice). KLK4 is secreted and lacks a transmembrane segment, while the antibody epitope is unspecified; use mild permeabilisation only when testing access to an intracellular epitope, and compare matched permeabilised and unpermeabilised sections (UniProt Q9Y5K2 topology; evidence payload: epitope unspecified; standard IF practice). Keep no-primary and single-channel controls to distinguish KLK4 signal from background and channel bleed-through (standard IF practice).
How can I reduce diffuse brown staining without losing KLK4 signal?
Check a no-primary control first; comparable brown staining there points to detection background rather than antibody-dependent KLK4 signal (standard IHC practice). For a peroxidase and DAB workflow, use a peroxide block, an appropriate protein block, and thorough washes, then change one condition at a time (standard IHC practice). The selected kidney image used antibody at 5 µg/mL; treat that as the image condition and titrate around it on matched sections rather than assuming it is optimal for every block (A03430 image caption; standard IHC practice). Recheck prostate glandular staining and tissue morphology after each adjustment so reduced background does not conceal a real signal (HPA: High in prostate glandular cells; standard IHC practice).
What is a defensible way to quantify KLK4 chromogenic staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define glandular cells as the primary scoring population for prostate sections, and record extracellular or luminal staining separately because KLK4 is secreted (HPA: High in prostate glandular cells; UniProt Q9Y5K2 subcellular annotation). For cellular signal, report the percentage of positive glandular cells and an H-score from 0–300, calculated from the percentages at intensity grades 0–3 (standard IHC practice). Normalise cell counts to the number of evaluable glandular cells, or extracellular signal to annotated glandular area in mm², using the same region rules across sections (standard IHC practice). Set thresholds against no-primary controls and keep scoring blinded to sample identity where feasible (standard IHC practice).
How do I distinguish true KLK4 positivity from staining artefacts?
Give greatest weight to reproducible glandular staining in prostate, where the tissue profile reports high signal in glandular cells (HPA: High in prostate glandular cells). KLK4 is secreted, so extracellular signal may be plausible, but isolated membrane outlines should not be treated as a required pattern (UniProt Q9Y5K2 topology). Inspect section edges, folds, necrotic areas and the no-primary control; staining confined to those features weakens a positive call (standard IHC practice). If brown signal persists without primary antibody, investigate endogenous peroxidase or detection background before attributing it to KLK4, and compare the result with a same-run prostate control (standard IHC practice; HPA: High in prostate glandular cells).
Boster reagents
Best KLK4 / Kallikrein-4 IHC Antibodies
A03430 has human IHC-P and IF validation, with images from human kidney tissue (catalog applications; IHC and IF image captions).
Real IHC data
Anti-Kallikrein-4 KLK4 Antibody
Cat # A03430
A03430 is listed for human IHC-P and IF (catalog applications; reactivity: Human). Its IHC image shows human kidney tissue at 5 μg/mL, and its IF image shows human kidney tissue at 20 μg/mL (catalog image captions).
Which to pick: Choose A03430 for human paraffin-section IHC (catalog: IHC-P; reactivity: Human); its kidney IHC caption reports 5 μg/mL but does not report the fixative (catalog IHC image caption). A03430 also has human IF validation, but ICC is not separately listed (catalog applications; reactivity: Human). No cross-species option is supported by this catalog payload (catalog reactivity: Human only).
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; tissue staining compartment unspecified (UniProt; 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 formalin fixation consistent across paraffin sections (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 A03430); Caveat: Secreted KLK4 may stain beyond producing cells (UniProt; HPA tissue IHC); Isoform / epitope: 2 isoforms; cleaved regions affect epitope choice (UniProt); Regulation: Prostate-enriched RNA expression (HPA tissue RNA). Evidence: UniProt Q9Y5K2, HPA tissue IHC · Enhanced, HPA ICC-IF · Secreted, Datasheet A03430, PMC13009279, PMC13201332. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/KLK4/immunohistochemistry-ihc-guide.