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GZMK / Granzyme K · IHC design guide
Design Immunohistochemistry for GZMK
Use 2 µg/ml of the catalog antibody for chromogenic IHC on paraffin sections (datasheet A06281-2). Compare cytoplasmic immune-cell staining with the tissue IHC profile and consider secretion when interpreting its distribution (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
GZMK 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 | Cytoplasm in immune cells (HPA tissue IHC) | |
| Staining pattern | Immune-cell cytoplasmic staining; texture unspecified (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A06281-2) | |
| Positive control | Appendix+3 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 sections (standard IHC practice; not target-specific) | |
| Caveat | Secretion may separate tissue RNA and protein patterns (HPA tissue IHC) | |
| Regulation | Constitutive secretion by CD8+ T cells (UniProt) | |
| Isoform / epitope | No isoforms; cleavage removes 1–26 from mature GZMK (UniProt) |
Section 1
Recommended GZMK IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A06281-2) is followed by published chromogenic GZMK IHC protocols for breast tissue (PMC11150209) and lung tissue (PMC13618230).
| Sample | Paraffin-embedded human spleen tissue; fixative not specified (datasheet A06281-2) |
| Fixation | Image fixative and duration unreported (datasheet A06281-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A06281-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A06281-2) |
| Primary antibody | Rabbit anti-GZMK, 2 μg/ml (datasheet A06281-2) |
| Primary incubation | Overnight at 4 °C (datasheet A06281-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A06281-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | GZMK-positive staining in lymphoid tissue of appendix (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in immune cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet: A06281-2); the published protocols used citrate retrieval (PMC11150209; PMC13618230).
Section 2
What Is the Expected GZMK Staining Pattern?
In paraffin-section IHC, expect mainly cytoplasmic staining in immune cells (HPA tissue IHC: cytoplasmic immune-cell profile). Granule-associated staining is plausible, and extracellular signal may reflect secretion (UniProt P49863: cytoplasmic granule; secreted). GZMK has no transmembrane segment (UniProt P49863 topology). HPA rates tissue staining Enhanced, with medium consistency between staining and RNA and an explicit secretion-related location caveat (HPA tissue IHC: reliability description).
| Discrete cytoplasmic staining in immune cells within appendix lymphoid tissue, lymph-node or tonsil germinal centers. | This fits the tissue profile; HPA reports medium staining at these sites (HPA tissue IHC: positive tissues). Morphology alone does not identify stained cells as CD8-positive T cells (UniProt P49863: CD8-positive T-cell specificity). |
| Predominantly nuclear staining, especially without a cytoplasmic immune-cell pattern. | Treat this as discordant with the reported tissue profile, then check counterstain, detection and a control section before interpreting it as GZMK (HPA tissue IHC: cytoplasmic immune-cell profile; standard IHC practice). |
| Strong staining of adipocytes or respiratory epithelium without the expected immune-cell pattern. | Investigate nonspecific binding or endogenous detection activity: these cell types were reported as not detected in the sampled tissues (HPA tissue IHC: adipose adipocytes; bronchus respiratory epithelium). |
| Diffuse color across tissue, including areas without distinct stained cells. | Secreted GZMK can complicate assignment of extracellular signal to its producing cell (UniProt P49863: secreted; HPA tissue IHC: secretion caveat). Widespread color also calls for checks of blocking, washing and detection controls (standard IHC practice). |
| No convincing signal in appendix lymphoid tissue or lymph-node germinal-center cells. | These are reported medium staining sites, so review section quality, retrieval, antibody conditions and detection with controls before calling the assay negative (HPA tissue IHC: positive tissues; standard IHC practice). |
💡Expected GZMK appearanceCall a slide positive when distinct cytoplasmic immune-cell staining is visible at an HPA-reported medium site; isolated nuclear or widespread uniform color is suspect (HPA tissue IHC: profile and positive tissues; standard IHC practice).
| Tissue choice and scoring | Appendix lymphoid tissue, lymph-node and tonsil germinal-center cells have reported medium staining; spleen red-pulp cells are reported low (HPA tissue IHC). Compare like cell populations rather than whole-section darkness (standard IHC practice). |
| Secretion and cell assignment | GZMK is granule-associated and secreted, including constitutive secretion by CD8-positive T cells without TCR stimulation (UniProt P49863: location note). An extracellular deposit need not mark the cell that made it (HPA tissue IHC: secretion caveat). |
| Topology and maturation | The precursor has a signal peptide at 1–24, a propeptide at 25–26 and a mature chain at 27–264; no transmembrane segment is annotated (UniProt P49863). The supplied record does not locate the antibody epitope. |
| Antibody evidence | HPA labels tissue IHC reliability Enhanced, while describing medium staining–RNA consistency and external characterization support (HPA tissue IHC: reliability description). Its IHC Enhanced entries are HPA063181 and HPA065895 (HPA antibodies). |
| Negative-cell comparison | HPA reports adipocytes in adipose tissue and respiratory epithelial cells in bronchus as not detected (HPA tissue IHC: negative tissues). Use their staining as a specificity warning, subject to section and detection controls (standard IHC practice). |
| IF/ICC Q: Should its reported Golgi pattern define the paraffin IHC readout? | A: No. HPA approves a Golgi location in ICC-IF, with images from JURKAT and REH; tissue IHC reports cytoplasmic immune-cell expression (HPA subcellular ICC-IF; HPA tissue IHC). Interpret each application against its own evidence. |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive-control tissue shows little or no staining. | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. | Check section integrity, retrieval, antibody application and detection with appropriate run controls (standard IHC practice). |
| Most cells stain uniformly, obscuring immune-cell boundaries. | Nonspecific antibody binding, incomplete washing or detection background may be contributing (standard IHC practice). | Inspect a negative reagent control and review blocking, antibody concentration, washes and chromogen development (standard IHC practice). |
| Brown signal appears in nuclei but little appears in cytoplasm. | The compartment conflicts with the HPA tissue IHC profile; counterstain or detection artifact may be involved (HPA tissue IHC: cytoplasmic immune-cell profile; standard IHC practice). | Compare with the counterstain and negative control, then reassess localization in a reported positive tissue (HPA tissue IHC: positive tissues; standard IHC practice). |
| Adipocytes or bronchial respiratory epithelial cells stain strongly. | Those sampled cell types were not detected by HPA; cross-reactivity or endogenous detection activity is possible (HPA tissue IHC: negative tissues; standard IHC practice). | Check negative reagent controls and the detection-system blocking step before assigning the color to GZMK (standard IHC practice). |
| An extracellular rim is scored as a GZMK-positive cell. | GZMK can be secreted, so deposited protein may be spatially separated from its source (UniProt P49863: secreted; HPA tissue IHC: secretion caveat). | Score cellular staining separately from extracellular staining and record the compartment seen (standard IHC practice). |
| Only faint red-pulp staining is seen in spleen. | HPA reports low staining in spleen red-pulp cells; weak signal there alone does not establish assay failure (HPA tissue IHC: low tissue). | Check an HPA-reported medium site in the same run before changing assay conditions (HPA tissue IHC: positive tissues; standard IHC practice). |
Experiment evidence
IHC & IF images showing GZMK staining
A06281-2 · IHCIHC analysis of Granzyme K/GZMK using anti-Granzyme K/GZMK antibody (A06281-2). Granzyme K/GZMK was detected in a paraffin-embedded section
A06281-1 · IHCImmunohistochemistry (IHC) analyzes of Granzyme K (H85) pAb in paraffin-embedded human breast carcinoma tissue at 1:100.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000113088-GZMK/tissue/cerebral+cortex#imid_20976007
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000113088-GZMK/tissue/colon#imid_20975805
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000113088-GZMK/tissue/liver#imid_20975806
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000113088-GZMK/tissue/kidney#imid_20975791
HPA · ICC-IF JURKATHuman Protein Atlas immunofluorescence, JURKAT, antibody HPA059953; green = GZMK, blue = nucleus, red = microtubules.
HPA · ICC-IF JURKATHuman Protein Atlas immunofluorescence, JURKAT, antibody HPA059953; green = GZMK, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for GZMK IHC & IF
🧪Run appendix first: its lymphoid tissue should stain for GZMK at a Medium level (HPA: Appendix, Lymphoid tissue, Medium). Use adipose tissue as the negative tissue because adipocytes are Not detected (HPA: Adipose tissue, Adipocytes, Not detected); on the appendix slide, use adjacent morphologically distinct cells to assess background, while interpreting any staining outside the lymphoid compartment cautiously because GZMK is secreted (UniProt P49863 subcellular).
Positive control tissue: Appendix (Lymphoid tissue, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show GZMK in JURKAT, REH, with annotated localisation: Golgi apparatus (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; an isotype control matched to the primary antibody’s host species, isotype, and monoclonal or polyclonal format; and a GZMK-knockout specimen as a biological negative if available (standard IHC practice). Quench endogenous peroxidase and check for residual DAB background in the appendix section (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported in the supplied evidence; the selected A06281-2 paraffin-section caption also does not report its fixative (selected-SKU tissue-IHC caption). The demonstrated IHC procedure used heat retrieval in EDTA, pH 8.0, followed by 2 μg/ml primary antibody overnight at 4°C, so retrieval is part of the documented starting protocol, but its necessity has not been established by a comparison (selected-SKU tissue-IHC caption). No supplied matched comparison establishes that frozen-section IHC or IF is easier; endogenous peroxidase in leukocyte-rich tissue can create chromogenic background, so assess it with the no-primary control (standard IHC practice).
HPA tissue IHC evidence for GZMK
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Secreted protein, tissue location of RNA and protein is expected to differ. External characterization data supports antibody staining.). 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 |
|---|---|---|---|---|
| Appendix | Lymphoid tissue | Medium | Protein (IHC) | HPA → |
| Lung | Alveolar cells type I | Medium | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Medium | Protein (IHC) | HPA → |
| Tonsil | Germinal center 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 → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced GZMK IHC Tips
Troubleshoot GZMK staining in paraffin sections by checking retrieval, cell identity, subcellular pattern, and controls before scoring signal.
Which retrieval condition should I start with for GZMK staining in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for the catalog antibody (datasheet A06281-2). The documented spleen section was stained after that retrieval, with 2 μg/ml primary antibody incubated overnight at 4°C (datasheet A06281-2). If immune-cell staining is weak, compare a shorter and longer heating interval on adjacent sections while holding detection and primary concentration constant (standard IHC practice). Record the heating method and cooling interval so a change in signal can be attributed to retrieval (standard IHC practice). Judge improvement by cleaner cytoplasmic staining in immune cells, the reported tissue pattern, rather than stronger staining everywhere (HPA tissue IHC: cytoplasmic expression in immune cells).
How should I troubleshoot suspected fixation effects on GZMK IHC?
The selected spleen caption identifies a paraffin section but does not state its fixative, so GZMK-specific fixation sensitivity is unknown (datasheet A06281-2). Record fixative, fixation duration, tissue thickness, and processing history before comparing sections, because these variables can change antigen accessibility (standard IHC practice). Run adjacent sections through the same EDTA pH 8.0 retrieval and 2 μg/ml primary incubation to isolate processing differences (datasheet A06281-2; standard IHC practice). If staining differs, repeat with matched processing and include a known staining section in the same run before assigning the difference to fixation (standard IHC practice). Do not infer fixation tolerance from reported tissue distribution (HPA tissue IHC).
What GZMK staining pattern should I expect within positive cells?
Assess cytoplasmic staining in immune cells first, since that is the reported tissue IHC pattern (HPA tissue IHC: cytoplasmic expression in immune cells). Granzyme K also has an annotated cytoplasmic-granule, cell-surface, and secreted distribution, with no transmembrane segment (UniProt P49863: subcellular location and topology). Its approved Golgi location comes from subcellular IF data and should be interpreted in that assay’s context (HPA subcellular: Golgi apparatus). In chromogenic sections, compare punctate or diffuse cytoplasmic signal against the 2 μg/ml spleen example and a matched negative control (datasheet A06281-2; standard IHC practice). Isolated nuclear staining warrants a specificity check because the supplied localisation records do not report a nuclear pool (UniProt P49863; HPA tissue IHC).
Could processing or epitope choice explain inconsistent GZMK staining?
GZMK has no annotated isoforms, so an isoform-specific tissue pattern is unsupported by this record (UniProt P49863: isoforms). Its precursor contains a signal sequence at residues 1–24, a propeptide at 25–26, and a mature chain at 27–264 (UniProt P49863: processing). The supplied antibody caption does not map its epitope, so accessibility of precursor and mature protein cannot be assumed equal (datasheet A06281-2). If results differ between preparations, compare matched sections under EDTA pH 8.0 retrieval and verify antibody specificity with appropriate controls (datasheet A06281-2; standard IHC practice). Do not attribute a staining shift to glycosylation; no glycosylation sites are annotated here (UniProt P49863: glycosylation).
How can IF help assess GZMK staining alongside this IHC result?
Use IF as a separate localisation check and multiplex GZMK with a CD8 marker, since CD8-positive T cells are the annotated expressing population (UniProt P49863: tissue specificity). Choose spectrally separated fluorophores, placing the weaker readout in a far-red channel when tissue autofluorescence affects shorter wavelengths (standard IF practice). Permeabilise when assessing intracellular granule or Golgi signal, and compare a non-permeabilised condition if testing accessible surface staining (UniProt P49863: subcellular location; HPA subcellular: Golgi apparatus; standard IF practice). Include single-colour and no-primary controls to assess bleed-through and background (standard IF practice). Compare cell identity and distribution with chromogenic IHC, while recognising that the paraffin spleen caption supplies no IF fixation or permeabilisation conditions (datasheet A06281-2).
How do I reduce diffuse or misleading brown signal?
First check a no-primary section and block endogenous peroxidase before HRP and DAB detection, separating tissue enzyme signal from antibody-dependent staining (standard IHC practice). The documented spleen staining used 10% goat serum blocking and an HRP-linked detection system with DAB (datasheet A06281-2). If background persists, titrate around the documented 2 μg/ml primary concentration, improve washes, and compare sections within the same run (datasheet A06281-2; standard IHC practice). Inspect tissue edges, folds, and damaged areas separately because they can retain reagents or stain unevenly (standard IHC practice). Accept a change only if immune-cell cytoplasmic signal remains distinct from surrounding background (HPA tissue IHC: cytoplasmic expression in immune cells).
What should I count when comparing GZMK IHC across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the analysis region and count viable, morphologically identifiable immune cells before scoring DAB signal (HPA tissue IHC: cytoplasmic expression in immune cells; standard IHC practice). Report the percentage of positive immune cells, or an H-score combining percentage and intensity, using the same threshold across sections (standard IHC practice). For spatial comparisons, report positive-cell density per mm² of viable tissue and separately record the immune-cell fraction (standard IHC practice). Normalise within comparable anatomical compartments, since reported staining includes low signal in splenic red pulp and medium signal in specified lymphoid regions (HPA tissue IHC). Exclude folds and necrotic areas consistently, and document the threshold with control sections (standard IHC practice).
How can I distinguish convincing GZMK positivity from artefact?
A convincing result places signal in identifiable immune-cell cytoplasm and reproduces across comparable sections (HPA tissue IHC: cytoplasmic expression in immune cells; standard IHC practice). Check CD8-rich cell identity where possible, since CD8-positive T cells are the annotated expressing population (UniProt P49863: tissue specificity). Treat isolated nuclear colour, broad staining at section edges, and staining confined to necrotic material as reasons to inspect controls and morphology (UniProt P49863: subcellular location; standard IHC practice). Compare a no-primary control after peroxidase blocking to identify endogenous enzyme signal in the DAB workflow (standard IHC practice). Interpret extracellular colour cautiously because GZMK is secreted and tissue RNA and protein locations may differ (UniProt P49863: subcellular location; HPA tissue IHC: reliability description).
Boster reagents
Best GZMK / Granzyme K IHC Antibodies
Anti-GZMK antibodies have IHC images from paraffin sections of human spleen and breast carcinoma (image captions); one also lists IF for Human, Mouse, and Rat (catalog: applications/reactivity).
Real IHC data
Anti-Granzyme K/GZMK Antibody
Cat # A06281-2
Real IHC data
Anti-Granzyme K (H85) GZMK Antibody
Cat # A06281-1
A06281-2 has an IHC image from a paraffin-embedded human spleen section (A06281-2 image caption). A06281-1 has an IHC image from paraffin-embedded human breast carcinoma and lists IF for Human, Mouse, and Rat (A06281-1 image caption; catalog: applications/reactivity).
Which to pick: For human tissue IHC, choose A06281-2 when its documented spleen workflow fits: EDTA retrieval, 2 μg/ml primary antibody, and DAB detection; the fixative is unreported (A06281-2 image caption). For IF or work across Human, Mouse, and Rat, choose A06281-1, which lists IF and IHC at 1:50–1:200 and has its own paraffin-section IHC image at 1:100 (catalog: applications/reactivity/dilutions; A06281-1 image caption). Both are described as polyclonal, while ICC validation is unreported (A06281-2 datasheet: Polyclonal; A06281-1 image caption: pAb; catalog: applications).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P49863 (GRAK_HUMAN, Granzyme K).
- Human Protein Atlas. GZMK tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. GZMK subcellular location (ICC-IF): Localized to the Golgi apparatus..
- Human Protein Atlas. GZMK antibody validation summary (3 antibodies).
- Exploring GZMK as a prognostic marker and predictor of immunotherapy response in breast cancer: unveiling novel insights into treatment outcomes. Journal of cancer research and clinical oncology 2024 — PMC11150209.
- Integrated single-cell TCR analysis across tissue identifies GZMK(+)CD8(+) T cells promoting fibrosis via TGF-β signalling in IPF. International journal of biological sciences 2026 — PMC13618230.
- Single-cell transcriptomic analysis reveals tumor cell heterogeneity and immune microenvironment features of pituitary neuroendocrine tumors. Genome medicine 2024 — PMC10759356.
- Intratumoral CD4(+) T Cells Mediate Anti-tumor Cytotoxicity in Human Bladder Cancer. Cell 2020 — PMC7321885.
- PubMed PMID:7758581 — UniProt-cited evidence.
- PubMed PMID:8656064 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for GZMK (Granzyme K). Expected localisation Cytoplasm in immune cells (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A06281-2). Validated IHC antibody: A06281-2 at 2 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Lymphoid tissue); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human spleen tissue; fixative not specified (datasheet A06281-2); 2. Fixation: Image fixative and duration unreported (datasheet A06281-2); 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: EDTA pH 8.0 (datasheet A06281-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A06281-2); 8. Primary antibody: Rabbit anti-GZMK, 2 μg/ml (datasheet A06281-2); 9. Primary incubation: Overnight at 4 °C (datasheet A06281-2). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Secretion may separate tissue RNA and protein patterns (HPA tissue IHC); Isoform / epitope: No isoforms; cleavage removes 1–26 from mature GZMK (UniProt); Regulation: Constitutive secretion by CD8+ T cells (UniProt). Evidence: UniProt P49863, HPA tissue IHC · Enhanced, HPA ICC-IF · Golgi apparatus., Datasheet A06281-2, PMC11150209, PMC13618230. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/GZMK/immunohistochemistry-ihc-guide.