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GZMH / Granzyme H · IHC design guide
Design Immunohistochemistry for GZMH
Plan paraffin-section IHC for GZMH using its cytolytic-granule location (UniProt) and staining in a subset of immune cells (HPA tissue IHC). The IHC-validated antibody has a 1:50–1:200 dilution range (datasheet); include a negative control (standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
GZMH 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 | Cytolytic granules expected (UniProt); tissue compartment unreported (HPA tissue IHC) | |
| Staining pattern | Staining in a subset of immune cells; compartment unreported (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Skin+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 | Presumed off-target staining can mislead (HPA tissue IHC) | |
| Regulation | Constitutively expressed in NK cells (UniProt) | |
| Isoform / epitope | 3 isoforms; chain 21–246; epitope map unknown (UniProt) |
Section 1
Recommended GZMH IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet) with the published GZMH tumor-tissue protocol (PMC13098063).
| Sample | Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A08559-1) |
| Fixation | Image fixative and duration unreported (datasheet A08559-1); 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-GZMH, 1:50-1:200 (datasheet A08559-1) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | GZMH-positive staining in cells in granular layer of skin (HPA tissue IHC: High). HPA tissue profile: Expression in a subset of immune cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval); the published protocol also uses citrate pH 6.0 for 20 min (PMC13098063).
Section 2
What Is the Expected GZMH Staining Pattern?
In paraffin-section IHC, expect GZMH in a subset of immune cells, with a cytoplasmic pattern compatible with cytolytic granules (HPA: tissue IHC; UniProt P20718: cytolytic granule). UniProt reports constitutive expression in NK cells and no transmembrane segment (UniProt P20718). HPA rates its tissue IHC evidence Enhanced, while noting medium agreement with RNA and presumed off-target staining that was disregarded (HPA: tissue IHC).
| Discrete cytoplasmic staining in scattered cells of lymph node or tonsil outside germinal centers, or in splenic red pulp. | This fits the reported distribution and medium staining in those sites (HPA: tissue IHC). Granule-compatible cytoplasmic signal is consistent with GZMH location (UniProt P20718). Morphology alone does not identify each stained cell as an NK cell; HPA calls the tissue pattern a subset of immune cells (HPA: tissue IHC). |
| Predominantly nuclear staining, or a continuous membrane outline, across many cells. | That pattern conflicts with the reported cytolytic-granule location and absence of a transmembrane segment (UniProt P20718). Treat it as suspect staining and compare with the expected tissue distribution and a reagent control (HPA: tissue IHC; general IHC practice). A secreted protein may appear beyond its producing cell, so location alone cannot prove an artefact (HPA: subcellular summary). |
| Broad staining of adipocytes or respiratory epithelium, instead of a restricted cell subset. | HPA reports GZMH as undetected in adipocytes of adipose tissue and respiratory epithelial cells of bronchus (HPA: tissue IHC). Check antibody cross-reactivity and detection chemistry before scoring such staining as GZMH (general IHC practice). HPA also reports that presumed off-target staining was disregarded in its assessment (HPA: tissue IHC). |
| A diffuse haze spans the section or obscures individual stained cells. | That cannot be scored reliably as the reported subset-of-immune-cells pattern (HPA: tissue IHC). Diffuse chromogenic background can arise from nonspecific reagent binding or detection activity (general IHC practice). Because HPA summarizes GZMH as secreted, extracellular signal is possible; a uniform haze still needs control-based evaluation (HPA: subcellular summary; general IHC practice). |
| No discernible staining in lymph node, spleen, or tonsil. | HPA reports medium staining in specified cells of these tissues, making them useful reference sections when the relevant regions are present (HPA: tissue IHC). Check tissue representation and IHC controls before calling the assay negative (general IHC practice). A whole-section negative result cannot establish absent expression in every immune cell (HPA: subset-of-immune-cells profile). |
💡Expected GZMH appearanceCall a section positive when discrete cytoplasmic signal marks a restricted cell subset in the reported lymphoid regions, consistent with cytolytic granules; HPA rates those sites medium, while broad nuclear, membrane, or uniform tissue staining is suspect (HPA: tissue IHC; UniProt P20718).
| Granule location and secretion | UniProt places GZMH in cytolytic granules; HPA summarizes it as secreted (UniProt P20718; HPA: subcellular summary). Interpret cell-associated cytoplasmic staining alongside morphology, while assessing any extracellular signal with controls (general IHC practice). |
| Tissue distribution and evidence strength | HPA reports medium staining in lymph node, spleen, and tonsil, and high staining in skin granular-layer cells (HPA: tissue IHC). Its Enhanced rating coexists with medium RNA agreement and disregarded presumed off-target staining; do not treat every stained cell as verified GZMH (HPA: tissue IHC). |
| Protein processing | UniProt annotates a signal peptide at residues 1–18, propeptide at 19–20, and mature chain at 21–246 (UniProt P20718). These boundaries matter when assessing a disclosed antibody epitope; no epitope is supplied here, so this record cannot predict which forms the antibody detects. |
| Isoforms and glycosylation | UniProt lists three isoforms and glycosylation sites at residues 71, 104, and 179 (UniProt P20718). Without an antibody epitope or isoform-specific validation, these annotations do not establish differences in IHC staining, antigen-retrieval needs, or fixation sensitivity. |
| Situation | Likely cause | Next action |
|---|---|---|
| Reference lymphoid tissue shows no signal. | The relevant cell subset may be absent from the sampled field, or assay sensitivity may be inadequate (HPA: tissue IHC; general IHC practice). | Inspect non-germinal-center areas in lymph node or tonsil, or splenic red pulp; verify a working IHC control and review antigen retrieval and antibody dilution using validated assay information (HPA: tissue IHC; general IHC practice). |
| Most nuclei or cell borders stain. | The distribution conflicts with granule localisation and lack of a transmembrane segment (UniProt P20718); nonspecific staining is possible (general IHC practice). | Compare a no-primary control and the reported restricted tissue pattern; review primary-antibody dilution and detection reagents before interpreting the stain (HPA: tissue IHC; general IHC practice). |
| Brown signal appears in cells HPA lists as undetected. | Antibody cross-reactivity or endogenous chromogenic detection activity may contribute (general IHC practice); HPA notes presumed off-target staining in its assessment (HPA: tissue IHC). | Check a no-primary control and the applicable endogenous-enzyme blocking step; compare cell identity and tissue distribution before assigning GZMH positivity (general IHC practice; HPA: tissue IHC). |
| Background masks scattered candidate-positive cells. | Nonspecific binding or detection background can obscure the restricted pattern reported by HPA (general IHC practice; HPA: tissue IHC). | Review blocking, washes, antibody dilution, and detection exposure against controls; score only cells whose signal remains distinguishable from background (general IHC practice). |
| Skin granular-layer staining dominates the interpretation. | HPA reports high staining there, but also cautions about medium RNA agreement and disregarded presumed off-target staining (HPA: tissue IHC). | Record the skin observation separately from the lymphoid-cell pattern and examine controls before assigning cell identity or target specificity (HPA: tissue IHC; general IHC practice). |
| Can this IHC pattern validate an IF/ICC result? | HPA provides no main subcellular location or ICC/IF image-bearing cell lines for this record (HPA: subcellular ICC/IF). | Use the separate IF/ICC guide for that application; assess fluorescence localisation and controls on their own terms, without treating this tissue IHC pattern as ICC/IF validation (HPA: subcellular ICC/IF; general IF practice). |
Experiment evidence
IHC & IF images showing GZMH staining
A08559-1 · IHCImmunohistochemistry (IHC) analyzes of Granzyme H (G75) pAb in paraffin-embedded human breast carcinoma tissue at 1:50.showing Secreted and
A08559-1 · IHCImmunohistochemistry (IHC) analyzes of Granzyme H (G75) pAb in paraffin-embedded human lung carcinoma tissue at 1:50.showing Secreted and Cy
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000100450-GZMH/tissue/cerebral+cortex#imid_7402767
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000100450-GZMH/tissue/colon#imid_7375382
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000100450-GZMH/tissue/liver#imid_7375375
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000100450-GZMH/tissue/kidney#imid_7375376
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for GZMH IHC & IF
🧪Run skin first and look for staining in cells of the granular layer (HPA: High in skin granular-layer cells). Use adipose tissue adipocytes as the negative comparison (HPA: Not detected in adipocytes); on the skin slide, cells outside the granular layer that lack specific granular staining provide an internal background reference, but their GZMH status should be verified independently (HPA: skin granular-layer cells High; UniProt P20718: cytolytic granule).
Positive control tissue: Skin (Cells in granular layer, 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 GZMH; derive a cell-line control from the positive tissue's cell type (Cells in granular layer) 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 species and clonality; use GZMH-knockout material, if available, as a biological specificity control (standard IHC practice). Block endogenous peroxidase, and assess endogenous biotin if using the caption’s avidin–biotin detection system (selected-SKU tissue-IHC caption: avidin-peroxidase).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state its fixative (selected-SKU tissue-IHC caption: A08559-1, 1:50, fixative not stated). Antigen-retrieval dependence is unreported, so retrieval conditions require empirical optimization (supplied target/application evidence: no retrieval conditions reported). The supplied evidence does not establish whether frozen sections or IF would be easier; in skin, pigment can complicate chromogenic interpretation (HPA: skin granular-layer cells High; standard IHC practice).
HPA tissue IHC evidence for GZMH
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. Presumed off target binding observed and disregarded.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Skin | Cells in granular layer | High | Protein (IHC) | HPA → |
| Lymph node | Non-germinal center cells | Medium | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Medium | Protein (IHC) | HPA → |
| Tonsil | Non-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 → |
| 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 GZMH IHC Tips
Troubleshoot GZMH staining in paraffin sections by checking retrieval, controls, cellular pattern, and scoring before assigning biological meaning.
How should I adjust retrieval when GZMH staining is weak or uneven?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval specification). If staining is weak, compare a modestly longer heating time on matched sections while keeping buffer, detection, and imaging conditions fixed (standard IHC practice). Include a lymph node or spleen section as a tissue comparison, since staining was reported in non-germinal center cells and red pulp cells, respectively (HPA tissue IHC). Compare signal with a primary-omission control before accepting a stronger retrieval condition (standard IHC practice). Excessive diffuse staining should prompt reassessment because GZMH is associated with cytolytic granules (UniProt P20718 subcellular annotation).
Could fixation explain loss of GZMH signal in paraffin sections?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (A08559-1 tissue-IHC caption). Record the fixative, fixation duration, tissue thickness, and processing history for each specimen so weak and strong cases can be compared fairly (standard IHC practice). Assess a known staining tissue alongside each processing batch; lymph node and spleen have reported GZMH-positive cell populations (HPA tissue IHC). If a batch loses signal, compare sections processed under documented conditions with the same retrieval and antibody settings (standard IHC practice). Do not attribute a difference to fixation until matched processing evidence supports that explanation (standard IHC practice).
What cellular pattern should count as plausible GZMH staining?
Look first for cytoplasmic granules in discrete cells, consistent with the cytolytic-granule annotation for GZMH (UniProt P20718 subcellular annotation). The selected paraffin-section image describes secreted and cytoplasmic granule staining in human breast carcinoma tissue (A08559-1 tissue-IHC caption). Extracellular signal therefore warrants inspection, but it should be distinguished from cellular staining and scored separately (A08559-1 tissue-IHC caption; standard IHC practice). GZMH has no annotated transmembrane segment, so a crisp, continuous membrane-only pattern needs independent support before interpretation (UniProt P20718 topology). Check serial sections and the primary-omission control when compartment boundaries are unclear (standard IHC practice).
How can isoforms or processing complicate GZMH epitope interpretation?
GZMH has 3 annotated isoforms, so document the antibody's stated immunogen or epitope before treating all staining as isoform equivalent (UniProt P20718 isoforms; standard IHC practice). The precursor carries a signal peptide at residues 1–18 and a propeptide at 19–20, while the annotated mature chain spans 21–246 (UniProt P20718 processing). Glycosylation sites are annotated at residues 71, 104, and 179, but their effect on this antibody's tissue staining is unreported (UniProt P20718 glycosylation; A08559-1 tissue-IHC caption). If specimens disagree, compare documented epitope coverage and processing rather than assuming an isoform change (standard IHC practice).
How should I investigate GZMH with multiplex IF alongside this IHC guide?
Treat IF as a separate assay requiring its own validation; the supplied antibody image documents paraffin-section chromogenic IHC (A08559-1 tissue-IHC caption). Pair GZMH with an independently validated NK-cell marker, because GZMH is constitutively expressed in NK cells (UniProt P20718 tissue specificity; standard IF practice). Choose spectrally separated fluorophores after checking unstained tissue for autofluorescence, and include single-stain controls for channel bleed-through (standard IF practice). Because GZMH is annotated in cytolytic granules and has no transmembrane segment, test permeabilisation for access to intracellular epitopes (UniProt P20718 subcellular annotation and topology; standard IF practice). Judge puncta against omission controls rather than transferring the chromogenic caption's settings directly to IF (standard IF practice).
How can I separate GZMH staining from chromogenic background?
Run a primary-omission section to identify signal generated by the detection system; the selected image used PBS instead of primary antibody for its negative control (A08559-1 tissue-IHC caption). Include a peroxidase block before chromogen development and examine endogenous enzyme activity if brown staining persists in the omission control (standard chromogenic IHC practice). Compare suspect deposits with tissue morphology and avoid counting pigment, folds, or damaged edges as positive cells (standard IHC practice). The tissue atlas reports presumed off-target binding that was disregarded, so unexpected cell populations need particular scrutiny (HPA tissue IHC reliability description). Recheck antibody concentration and blocking conditions if background rises across otherwise matched sections (standard IHC practice).
What should I score when GZMH-positive cells are sparse? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue compartment and count discrete GZMH-positive cells per mm² of viable tissue, recording the sampled area and exclusion rules (standard IHC practice). For a defined cell population, report the percentage positive and, when intensity is reproducible, an H-score from 0–300 (standard IHC practice). Keep cytoplasmic granule staining separate from extracellular deposits, since both patterns appear in the selected paraffin-section caption (A08559-1 tissue-IHC caption). Normalise counts to viable tissue area or the total eligible cells in the same compartment, and apply one threshold across batches (standard IHC practice). Report replicate fields and observer agreement when scoring sparse populations (standard IHC practice).
When does a GZMH-positive section support a biological conclusion?
Give greatest weight to discrete cells with cytoplasmic granule staining in an appropriate immune-cell context (UniProt P20718 subcellular and NK-cell annotations; HPA tissue IHC profile). Compare lymph node or spleen patterns with an omission control; the atlas reports staining in non-germinal center and red pulp cells but also notes presumed off-target binding (HPA tissue IHC). Review membrane-only staining, necrotic areas, and section edges before accepting a positive result (UniProt P20718 topology; standard IHC practice). Persistent brown signal without primary antibody raises concern for endogenous enzyme activity or detection background (standard chromogenic IHC practice). Treat extracellular staining separately, since GZMH is also described as secreted (HPA subcellular summary).
Boster reagents
Best GZMH / Granzyme H IHC Antibodies
A08559-1 is listed for IHC and IF in human, mouse, and rat (catalog: applications/reactivity); its pictured IHC uses paraffin-embedded human breast carcinoma (IHC image caption).
Real IHC data
Anti-Granzyme H (G75) GZMH Antibody
Cat # A08559-1
A08559-1 is listed for IHC and IF in human, mouse, and rat (catalog: applications/reactivity). Its IHC captions show paraffin-embedded human breast and lung carcinoma at 1:50; no IF image is supplied (IHC image captions; catalog: IF images).
Which to pick: For tissue IHC, choose A08559-1, a rabbit pAb pictured on paraffin-embedded human breast and lung carcinoma; the captions do not report the fixative (catalog: host; IHC image captions). For IF, A08559-1 is listed at 1:50–1:200, but ICC is unlisted and no IF image is supplied (catalog: applications, IF dilution, IF images). For mouse or rat samples, A08559-1 lists reactivity with both species, while its supplied IHC images show human tissue only (catalog: reactivity; IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P20718 (GRAH_HUMAN, Granzyme H).
- Human Protein Atlas. GZMH tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. GZMH subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. GZMH antibody validation summary (1 antibodies).
- Blood Based Biomarkers for Predicting Treatment Response to Immune Checkpoint Inhibitors After EGFR-TKI Resistance in Non-Small Cell Lung Cancer. Thoracic cancer 2026 — PMC13098063.
- Granzyme h serum levels variations with both reproductive hormone receptors, and related hormone receptors in breast cancer patients. Iranian journal of cancer prevention 2014 — PMC4142953.
- PubMed PMID:2193684 — UniProt-cited evidence.
- PubMed PMID:2049336 — UniProt-cited evidence.
- PubMed PMID:2402757 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for GZMH (Granzyme H). Expected localisation Cytolytic granules expected (UniProt); tissue compartment unreported (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A08559-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Skin (Cells in granular layer); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A08559-1); 2. Fixation: Image fixative and duration unreported (datasheet A08559-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-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-GZMH, 1:50-1:200 (datasheet A08559-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Presumed off-target staining can mislead (HPA tissue IHC); Isoform / epitope: 3 isoforms; chain 21–246; epitope map unknown (UniProt); Regulation: Constitutively expressed in NK cells (UniProt). Evidence: UniProt P20718, HPA tissue IHC · Enhanced, HPA ICC-IF · Secreted, Datasheet A08559-1, PMC13098063. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/GZMH/immunohistochemistry-ihc-guide.