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GZMM / Granzyme M · IHC design guide
Design Immunohistochemistry for GZMM
Plan GZMM paraffin IHC with the catalog antibody at 2–5 μg/ml (datasheet A08286-2). Bone marrow hematopoietic cells and spleen red pulp cells show high staining, while secretion may complicate interpretation of tissue location (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
GZMM Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic granules or secreted; tissue location unconfirmed (UniProt) | |
| Staining pattern | High in a subset of immune cells; compartment unreported (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A08286-2) | |
| Positive control | Bone marrow+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A08286-2) | |
| Caveat | Secretion may separate tissue staining from RNA expression (HPA tissue IHC) | |
| Regulation | Constitutive in activated NK cells (UniProt) | |
| Isoform / epitope | No isoforms annotated; epitopes in aa 1–25 may be lost (UniProt) |
Section 1
Recommended GZMM IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet A08286-2) with published GZMM staining in endometrial cancer (PMC10792215) and colon carcinoma (PMC4467404).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A08286-2) |
| Fixation | Image fixative and duration unreported (datasheet A08286-2); 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 A08286-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A08286-2) |
| Primary antibody | Rabbit anti-GZMM, 2-5 μg/ml (datasheet A08286-2) |
| Primary incubation | Overnight at 4 °C (datasheet A08286-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A08286-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | GZMM-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: High expression in subset of immune cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet A08286-2); neither included article specifies retrieval conditions (PMC10792215; PMC4467404).
Section 2
What Is the Expected GZMM Staining Pattern?
GZMM is a secreted protein associated with cytoplasmic granules of large granular lymphocytes and has no transmembrane segment (UniProt P51124). Expect staining in a subset of immune cells, consistent with its expression in activated NK cells (HPA tissue IHC: Enhanced; UniProt P51124). Tissue location can be difficult to interpret for a secreted protein, and HPA disregarded presumed off-target staining in its assessment (HPA tissue IHC: reliability description).
| Distinct cytoplasmic granules in scattered immune cells, with strong staining among bone marrow hematopoietic cells or splenic red-pulp cells. | This fits the reported granule location and tissue pattern (UniProt P51124; HPA tissue IHC: High in bone marrow hematopoietic cells and splenic red-pulp cells). Cell shape alone does not establish NK identity; use an appropriate cell marker if that distinction matters (general IHC practice). |
| Strong, uniform nuclear or sharply membrane-bound staining dominates the section. | That pattern is inconsistent with the annotated granule and secreted locations and absence of a transmembrane segment (UniProt P51124). Treat it as suspect until controls and a second detection approach support it (general IHC practice). |
| Strong staining appears in adipocytes or bronchial respiratory epithelium. | HPA reports GZMM as not detected in those cells, so investigate nonspecific binding or endogenous detection activity (HPA tissue IHC: adipocytes and bronchial respiratory epithelial cells not detected; general IHC practice). Do not apply this rule to every nonimmune cell: HPA reports medium staining in alveolar type I and tonsillar squamous epithelial cells (HPA tissue IHC). |
| Diffuse color covers many cell types or the section background, obscuring individual positive cells. | The result cannot be scored as the reported subset-of-immune-cells pattern (HPA tissue IHC: profile). Check the reagent control, detection system and wash steps for background, and compare with a known-positive tissue processed in the same run (general IHC practice). |
| No staining is visible in bone marrow hematopoietic cells or splenic red-pulp cells. | These are reported high-staining locations, so an entirely blank known-positive control calls the assay result into question (HPA tissue IHC: High). Check antibody and detection performance before interpreting an unstained study specimen as GZMM-negative (general IHC practice). |
💡Expected GZMM appearanceCall a result positive when discrete cytoplasmic staining occurs in a subset of cells, especially at HPA high-staining immune-cell locations; diffuse field-wide or dominant nuclear staining is suspect (UniProt P51124 granules; HPA tissue IHC: profile and High locations).
| Compartment and secretion | Granule-associated cytoplasmic signal is expected, but extracellular signal may require cautious interpretation because GZMM is also secreted (UniProt P51124). HPA warns that RNA and protein tissue locations may differ for a secreted variant (HPA tissue IHC: reliability description). |
| Protein processing and epitope | UniProt annotates a signal peptide at residues 1–23, propeptide at 24–25 and mature chain at 26–257 (UniProt P51124). If an antibody's epitope is known, compare it with these regions when interpreting staining; no epitope is supplied here (general IHC practice). |
| Antibody validation and tissue comparison | The listed antibody HPA015624 has Enhanced IHC validation (HPA antibodies: HPA015624). Use the reported High bone marrow or spleen locations alongside a reported not-detected cell population to judge whether the observed pattern is selective (HPA tissue IHC). |
| IF/ICC: Is a cellular staining pattern established? | HPA lists GZMM as secreted but provides no main subcellular location or ICC-IF cell-line images (HPA subcellular). Granule localisation is annotated by UniProt, so an IF/ICC result needs independent validation before assigning a precise cellular pattern (UniProt P51124; general IF practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive bone marrow or spleen control is blank. | The run may have insufficient primary-antibody or detection signal; those locations are reported High by HPA (HPA tissue IHC; general IHC practice). | Verify the control section, primary-antibody step and detection reagents; review antigen-retrieval settings as a general IHC workflow check (general IHC practice). No GZMM-specific retrieval condition is supplied. |
| Study specimen is blank while the positive control stains. | The specimen may contain few expressing cells; GZMM staining is reported in a subset of immune cells (HPA tissue IHC: profile). | Review an appropriate immune-rich region and score the relevant cells rather than the entire section; document the positive control in the same run (HPA tissue IHC: profile; general IHC practice). |
| Nuclear or membrane signal overwhelms cytoplasmic staining. | The dominant compartment conflicts with the granule and secreted annotations and lack of a transmembrane segment (UniProt P51124). | Inspect the reagent control and repeat with a validated detection setup; avoid calling the compartment GZMM-specific without corroboration (general IHC practice). |
| Color appears broadly in cells HPA reports as not detected. | Nonspecific binding or endogenous detection activity is possible (general IHC practice); HPA reports no staining in adipocytes and bronchial respiratory epithelial cells (HPA tissue IHC). | Compare reagent controls and examine the detection-blocking step; assess the named cell population, since HPA also reports medium staining in some nonimmune cells (general IHC practice; HPA tissue IHC). |
| Background obscures scattered positive cells. | Broad reagent or detection background can prevent assessment of the reported subset pattern (general IHC practice; HPA tissue IHC: profile). | Compare a no-primary control, review blocking and washes, and rescore only when individual stained cells can be distinguished (general IHC practice). |
| A second tissue gives a different staining intensity. | HPA reports High staining in bone marrow hematopoietic and splenic red-pulp cells, but Medium staining in lymph-node non-germinal-center cells (HPA tissue IHC). | Compare the same annotated cell populations and run controls before treating an intensity difference as assay failure (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing GZMM staining
A08286-2 · IHCIHC analysis of Granzyme M/GZMM using anti-Granzyme M/GZMM antibody (A08286-2). Granzyme M/GZMM was detected in a paraffin-embedded section
A08286-2 · IHCIHC analysis of Granzyme M/GZMM using anti-Granzyme M/GZMM antibody (A08286-2). Granzyme M/GZMM was detected in a paraffin-embedded section
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000197540-GZMM/tissue/cerebral+cortex#imid_4021180
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000197540-GZMM/tissue/colon#imid_4021090
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000197540-GZMM/tissue/liver#imid_4021107
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000197540-GZMM/tissue/kidney#imid_4021100
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for GZMM IHC & IF
🧪Run spleen first: cells in the red pulp should stain (HPA: High in cells in red pulp). Use adipose tissue as the negative tissue, where adipocytes should lack staining (HPA: Not detected in adipocytes); on the spleen slide, use cells without specific staining as internal negative comparators rather than assuming every red-pulp cell is positive (HPA: High in cells in red pulp).
Positive control tissue: Bone marrow (Hematopoietic 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 GZMM; derive a cell-line control from the positive tissue's cell type (Hematopoietic cells) and confirm it by RNA or western blot first.
Technical controls: Include no-primary (secondary-only), a concentration-matched rabbit IgG isotype control for the catalog antibody, and a GZMM-knockout specimen as a biological negative when available (selected-SKU IHC caption: rabbit primary; standard IHC control practice). Quench endogenous peroxidase and inspect spleen sections for pigment or blood-associated DAB background before scoring (selected-SKU IHC caption: HRP/DAB; standard chromogenic IHC practice).
⚠️Feasibility: The selected-SKU paraffin-section image used heat retrieval in EDTA at pH 8.0, but the caption does not establish whether retrieval is required; its fixative is unreported, and no target-specific fixation window or effect is supplied (selected-SKU IHC caption: EDTA retrieval; fixative not stated). Frozen-section or IF/ICC feasibility cannot be ranked against paraffin IHC from these data, and HPA lists no ICC-IF cell-line images (HPA subcellular: no ICC-IF cell-line images). In spleen, blood-associated pigment or residual peroxidase can complicate chromogenic interpretation (standard chromogenic IHC practice).
HPA tissue IHC evidence for GZMM
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex. Presumed off target binding observed and disregarded. Antibody staining in cells/structures not annotated, view images.). 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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | High | Protein (IHC) | HPA → |
| Lung | Alveolar cells type I | Medium | Protein (IHC) | HPA → |
| Lymph node | Non-germinal center cells | Medium | Protein (IHC) | HPA → |
| Tonsil | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced GZMM IHC Tips
Troubleshoot GZMM chromogenic IHC by checking retrieval, tissue processing, cellular localisation, controls and scoring before interpreting weak or unexpected staining.
What retrieval should I try first for GZMM paraffin-section IHC?
Start with heat-mediated EDTA at pH 8.0 for GZMM paraffin-section IHC (datasheet A08286-2). The selected image used this retrieval before 2 μg/ml primary antibody overnight at 4°C; keep antibody and detection conditions constant while assessing retrieval (datasheet A08286-2). If staining is weak, compare heating times on matched sections and document the temperature, cooling interval and any tissue damage (standard IHC practice). Include a positive bone marrow section and a no-primary control to distinguish weak retrieval from nonspecific DAB deposition (HPA: high staining in bone marrow hematopoietic cells; standard IHC practice).
Could fixation explain inconsistent GZMM staining between paraffin sections?
The selected paraffin-section caption does not report its fixative, so GZMM-specific fixation sensitivity is unknown (datasheet A08286-2). Record the fixative, fixation duration, specimen thickness and delay before fixation for each sample being compared (standard IHC practice). On matched sections, hold EDTA retrieval at pH 8.0 and primary antibody at 2 μg/ml while investigating processing differences (datasheet A08286-2; standard IHC practice). Include a positive control and no-primary control in each run to assess signal loss and detection background (standard IHC practice). HPA staining patterns and the annotated glycosylation site at residue 177 do not establish how any fixative affects this epitope (HPA: tissue IHC; UniProt P51124 glycosylation).
What cellular pattern supports GZMM staining in tissue IHC?
Assess punctate cytoplasmic staining in candidate large granular lymphocytes first, because GZMM is annotated in their cytoplasmic granules (UniProt P51124 localisation). GZMM is also annotated as secreted and lacks a transmembrane segment, so do not require a crisp cell-surface rim (UniProt P51124 localisation and topology). Compare the stained cells with their morphology and nearby immune infiltrate on the counterstained section (standard IHC practice). Diffuse extracellular colour alone cannot identify the producing cell; check a no-primary section and examine whether colour follows tissue folds or damaged areas (UniProt P51124 secretion; standard IHC practice). Record granular cellular signal separately from extracellular staining when scoring cases (standard IHC practice).
How can processing or epitope location affect GZMM IHC interpretation?
GZMM has no annotated isoforms in the supplied record, so an apparent staining difference should not be assigned to an isoform without further evidence (UniProt P51124 isoforms). Its signal peptide spans residues 1–23, propeptide 24–25, and annotated mature chain 26–257 (UniProt P51124 processing). A glycosylation site is annotated at residue 177, but the catalog antibody's epitope is not specified in the supplied caption (UniProt P51124 glycosylation; datasheet A08286-2). Compare retrieval conditions on adjacent sections while holding detection constant, and confirm unexpected staining with an independently characterised antibody if available (standard IHC practice).
How should I investigate GZMM localisation with companion IF?
Use companion IF to compare GZMM signal with a marker for the expected NK-cell population, since GZMM is highly expressed in activated NK cells (UniProt P51124 tissue specificity). Choose fluorophores after examining unstained tissue for autofluorescence, and include single-colour controls before interpreting overlap (standard IF practice). For an antibody recognising an intracellular epitope, assess permeabilisation because GZMM is annotated in cytoplasmic granules and has no transmembrane segment (UniProt P51124 localisation and topology; standard IF practice). The supplied IHC caption establishes 2 μg/ml for paraffin-section chromogenic detection, but supplies no IF dilution or IF fixation evidence (datasheet A08286-2). Optimise those IF conditions separately (standard IF practice).
How can I reduce diffuse brown background in GZMM IHC?
Start by comparing the stained section with a no-primary control and checking whether colour follows tissue edges, folds or damaged regions (standard IHC practice). The selected paraffin-section image used 10% goat serum blocking, 2 μg/ml primary antibody and DAB detection; those settings provide a documented starting point for this catalog antibody (datasheet A08286-2). Block endogenous peroxidase before HRP detection and wash thoroughly between incubations to limit nonspecific colour (standard IHC practice). If background persists, titrate primary antibody on matched sections while keeping EDTA retrieval at pH 8.0 and exposure to DAB consistent (datasheet A08286-2; standard IHC practice).
How should I score sparse GZMM-positive cells in paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the viable tissue area and immune-cell compartment before scoring, then apply the same selection rules across cases (standard IHC practice). Report GZMM-positive cells as a percentage of counted immune cells or as positive-cell density per mm² of viable tissue; state the denominator used (standard IHC practice). If using an H-score, document intensity categories and the percentage of cells in each category, and specify whether granular staining qualifies as positive (standard IHC practice). Normalise comparisons to immune-cell abundance because HPA reports high expression in a subset of immune cells, while GZMM is highly expressed in activated NK cells (HPA: tissue IHC profile; UniProt P51124 tissue specificity).
When should unexpected GZMM staining be treated cautiously?
Give greatest weight to staining in plausible immune cells with cytoplasmic granularity, consistent with GZMM's large granular lymphocyte granules and activated NK-cell expression (UniProt P51124 localisation and tissue specificity). Treat isolated nuclear staining or uniform epithelial colour cautiously, and compare those findings with morphology, a no-primary control and an independently characterised antibody when available (UniProt P51124 localisation; standard IHC practice). Review edge staining, necrotic areas and endogenous peroxidase activity before calling DAB-positive material true antigen signal (standard IHC practice). HPA explicitly notes presumed off-target binding and staining in unannotated structures, so its reported tissue patterns alone cannot resolve an unexpected cell identity (HPA: reliability description).
Boster reagents
Best GZMM / Granzyme M IHC Antibodies
One anti-GZMM antibody has real IHC data from human paraffin-embedded breast and colon cancer sections; no IF figure is supplied (A08286-2 image captions; catalog: IF images).
Real IHC data
Anti-Granzyme M/GZMM Antibody
Cat # A08286-2
A08286-2 is listed for human IHC at 2–5 μg/ml (catalog: applications, reactivity, IHC dilution). Its own IHC captions show paraffin-embedded human breast and colon cancer sections stained at 2 μg/ml (A08286-2 image captions).
Which to pick: Choose A08286-2 for human paraffin-section IHC: it is a rabbit polyclonal antibody listed for human IHC, with breast and colon cancer section images (catalog: host, clonality, applications, reactivity; A08286-2 image captions). No SKU in the supplied catalog is listed for IF/ICC or species beyond human (catalog: applications, reactivity). The A08286-2 captions report paraffin embedding but leave the fixative unreported (A08286-2 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P51124 (GRAM_HUMAN, Granzyme M).
- Human Protein Atlas. GZMM tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. GZMM subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. GZMM antibody validation summary (1 antibodies).
- Identification of the prognostic immune subtype in copy-number high endometrial cancer. Journal of gynecologic oncology 2024 — PMC10792215.
- Granzyme M expressed by tumor cells promotes chemoresistance and EMT in vitro and metastasis in vivo associated with STAT3 activation. Oncotarget 2015 — PMC4467404.
- Concentration of non-myocyte proteins in arterial media of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. PloS one 2023 — PMC9907840.
- PubMed PMID:8245461 — UniProt-cited evidence.
- PubMed PMID:7713495 — UniProt-cited evidence.
- PubMed PMID:15057824 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for GZMM (Granzyme M). Expected localisation Cytoplasmic granules or secreted; tissue location unconfirmed (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A08286-2). Validated IHC antibody: A08286-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A08286-2); 2. Fixation: Image fixative and duration unreported (datasheet A08286-2); 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 A08286-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A08286-2); 8. Primary antibody: Rabbit anti-GZMM, 2-5 μg/ml (datasheet A08286-2); 9. Primary incubation: Overnight at 4 °C (datasheet A08286-2). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A08286-2); Caveat: Secretion may separate tissue staining from RNA expression (HPA tissue IHC); Isoform / epitope: No isoforms annotated; epitopes in aa 1–25 may be lost (UniProt); Regulation: Constitutive in activated NK cells (UniProt). Evidence: UniProt P51124, HPA tissue IHC · Enhanced, HPA ICC-IF · Secreted, Datasheet A08286-2, PMC10792215, PMC4467404. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/GZMM/immunohistochemistry-ihc-guide.