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GZMA / Granzyme A · IHC design guide
Design Immunohistochemistry for GZMA
Plan chromogenic GZMA IHC-P around the cytoplasmic tissue pattern (HPA tissue IHC), using human tonsil as a positive control (HPA tissue IHC; datasheet: PA1588 IHC-P image). Start the catalog antibody at 0.5–1 μg/mL (datasheet: PA1588) and compare staining with matched controls.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
GZMA 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 staining in tissue (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic signal in several tissues, including immune cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Appendix+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 PA1588) | |
| Caveat | Secreted GZMA may stain away from its RNA source (HPA tissue IHC) | |
| Regulation | Cytotoxic T/NK cell content affects signal (UniProt) | |
| Isoform / epitope | 2 isoforms; check epitope against mature chain 29–262 (UniProt) |
Section 1
Recommended GZMA IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published GZMA IHC protocols for salivary gland and premotor cortex sections (PMC10710186; PMC11555691).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet PA1588); 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-GZMA, 0.5-1μg/ml (datasheet PA1588) |
| 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 | GZMA-positive staining in lymphoid tissue of appendix (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in several tissues, including immune cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen-retrieval rule); the salivary gland protocol also uses citrate pH 6.0 (PMC10710186).
Section 2
What Is the Expected GZMA Staining Pattern?
GZMA is a secreted protein stored in cytoplasmic granules of cytotoxic T cells and NK cells; it has no transmembrane segment (UniProt P12544). In tissue IHC, expect cytoplasmic staining among immune cells, with Medium staining reported in selected lymphoid and hematopoietic compartments (HPA tissue IHC). HPA rates the tissue staining reliability Enhanced, while noting low agreement between antibody staining and RNA expression because secreted protein can appear away from its site of production (HPA tissue IHC).
| Discrete cytoplasmic staining in immune cells of appendix lymphoid tissue, bone marrow hematopoietic cells, lymph node or tonsil germinal center cells, or splenic red pulp cells. | This matches the compartments where HPA reports Medium tissue staining (HPA tissue IHC). A compatible cell and compartment support the expected pattern, but morphology alone cannot establish that each stained cell is a cytotoxic T cell or NK cell (UniProt P12544; standard IHC interpretation). |
| Predominantly nuclear staining, or a crisp membrane outline instead of cytoplasmic staining. | Treat this as a pattern mismatch: HPA describes cytoplasmic tissue expression, and UniProt places GZMA in cytoplasmic granules or outside the cell, with no transmembrane segment (HPA tissue IHC; UniProt P12544). Check whether counterstain, precipitate, or nonspecific detection could explain the apparent compartment (standard IHC practice). |
| Strong signal in adipocytes or bronchial respiratory epithelial cells while the expected immune-cell pattern is absent. | HPA reports GZMA as Not detected in those listed cell types (HPA tissue IHC). Consider antibody cross-reactivity or endogenous chromogenic detection activity before assigning the signal to GZMA; compare the staining with a no-primary control (standard IHC practice). |
| Diffuse color across tissue, including cells expected to be negative, with little cell-specific contrast. | A widespread haze does not resemble HPA's cytoplasmic pattern in several tissues, including immune cells (HPA tissue IHC). Possible technical sources include insufficient blocking, excessive antibody concentration, or detection background; these are general IHC possibilities, not documented GZMA-specific effects (standard IHC practice). |
| No cytoplasmic signal in a section containing one of HPA's Medium-staining compartments. | A negative result there is inconclusive until section quality and detection are checked: HPA reports staining in the specified compartments, but does not establish that every specimen or cell will stain (HPA tissue IHC). Verify the anatomical compartment and assess a positive-control section and the antibody's IHC suitability (standard IHC practice; HPA antibodies). |
💡Expected GZMA appearanceCall a result compatible when immune cells show discernible cytoplasmic staining in an HPA-reported Medium compartment; isolated nuclear or membrane staining, or dominant staining of HPA-listed negative cell types, is a pattern mismatch requiring controls (HPA tissue IHC; UniProt P12544; standard IHC interpretation).
| Subcellular distribution | GZMA is stored in cytoplasmic granules, is secreted, and can be delivered into target cells by perforin (UniProt P12544). Interpret cell-associated cytoplasmic signal in its tissue context; extracellular signal alone cannot identify the producing cell (UniProt P12544; standard IHC interpretation). |
| Choice of tissue and compartment | HPA reports Medium staining in specified appendix, bone marrow, lymph node, spleen, and tonsil compartments, but Not detected staining in listed adipocytes and bronchial epithelium (HPA tissue IHC). Score the annotated cells within each tissue instead of treating the whole section as uniformly positive or negative (standard IHC practice). |
| Antibody validation | HPA054134 is IHC Approved; HPA076751 is IHC Enhanced, meaning its pattern is supported by at least two independent antibodies or orthogonal data (HPA antibodies). These labels inform confidence in an observed pattern; they do not prove that every signal in a new specimen is specific (HPA antibodies; standard IHC interpretation). |
| Processing and epitope uncertainty | UniProt lists a signal peptide at residues 1–26, a propeptide at 27–28, a mature chain at 29–262, and one glycosylation site at residue 170 (UniProt P12544). The payload gives no antibody epitope, so it cannot establish which processed form an IHC antibody recognizes (UniProt P12544; HPA antibodies). |
| Retrieval and detection | Follow the chosen IHC-validated antibody's documented paraffin-section workflow when available; optimize retrieval, antibody concentration, blocking, and chromogenic detection against controls as general IHC practice. Neither HPA tissue staining nor UniProt topology establishes a GZMA-specific fixation or retrieval effect (HPA tissue IHC; UniProt P12544; standard IHC practice). |
| IF/ICC Q: Can this IHC pattern define an IF/ICC protocol? | No ICC-IF images or main cellular location are available in the supplied HPA subcellular record, and neither listed HPA antibody has an ICC validation entry (HPA subcellular; HPA antibodies). The IHC tissue pattern supports an expectation of cytoplasmic signal, but does not establish IF/ICC conditions or validate an IF/ICC result (HPA tissue IHC; standard assay interpretation). |
| Situation | Likely cause | Next action |
|---|---|---|
| A proposed positive control has no staining. | The sampled area may lack the specific HPA-reported positive compartment, or the staining run may have failed; a negative section alone cannot distinguish these explanations (HPA tissue IHC; standard IHC practice). | Confirm that the section contains an HPA-listed Medium compartment, then review section integrity, the IHC-validated antibody instructions, and a working positive-control section (HPA tissue IHC; HPA antibodies; standard IHC practice). |
| Nuclei dominate the positive signal. | Predominantly nuclear staining conflicts with the reported cytoplasmic tissue pattern and cytoplasmic granule location (HPA tissue IHC; UniProt P12544). Counterstain or nonspecific detection may complicate interpretation (standard IHC practice). | Inspect the signal before and after counterstaining where feasible, compare with a no-primary control, and repeat using the chosen antibody's IHC conditions if the mismatch persists (standard IHC practice; HPA antibodies). |
| Adipocytes or bronchial respiratory epithelium stain strongly. | Those cell types are Not detected in the supplied HPA tissue record; cross-reactivity or endogenous chromogenic activity is possible (HPA tissue IHC; standard IHC practice). | Compare a no-primary control and a positive-compartment section; address endogenous detection activity and reassess antibody concentration if controls implicate technical staining (HPA tissue IHC; standard IHC practice). |
| The entire section shows weak, diffuse color. | Background from blocking, antibody concentration, washing, or chromogenic detection can obscure a cell-specific pattern (standard IHC practice). HPA's reported cytoplasmic staining does not establish which technical step caused the haze (HPA tissue IHC). | Check a no-primary control, then optimize blocking, washing, antibody concentration, and detection while preserving a positive tissue control for comparison (standard IHC practice; HPA tissue IHC). |
| Protein staining and local RNA expectations disagree. | HPA explicitly notes low consistency between antibody staining and RNA expression data because secreted protein may be located away from the tissue site of its RNA (HPA tissue IHC). | Record the stained cell type and compartment, compare them with HPA's tissue observations, and avoid calling the IHC result false solely from local RNA abundance (HPA tissue IHC; standard IHC interpretation). |
| Granular cytoplasmic staining is hard to distinguish from nearby extracellular color. | UniProt describes both cytoplasmic granule storage and secretion, so location alone may not identify the cell producing the protein (UniProt P12544). Chromogenic spread or background can further blur boundaries (standard IHC practice). | Use the counterstain to identify cell borders, compare negative and no-primary controls, and score convincing cell-associated cytoplasmic staining separately from extracellular color (UniProt P12544; standard IHC practice). |
Experiment evidence
IHC & IF images showing GZMA staining
PA1588 · IHCAnti-Granzyme A antibody, PA1588, IHC(P) IHC(P): Human Tonsil Tissue
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000145649-GZMA/tissue/cerebral+cortex#imid_20851183
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000145649-GZMA/tissue/colon#imid_20851117
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000145649-GZMA/tissue/liver#imid_20851118
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000145649-GZMA/tissue/kidney#imid_20851127
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for GZMA IHC & IF
🧪Run tonsil first and assess staining in germinal center cells (HPA: Medium in tonsil germinal center cells; PA1588 IHC(P) caption: human tonsil). Use bronchus respiratory epithelial cells as the negative tissue (HPA: Not detected in bronchus respiratory epithelial cells); cells without specific staining on the tonsil slide should show only background (standard IHC practice).
Positive control tissue: Appendix (Lymphoid tissue, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for GZMA; derive a cell-line control from the positive tissue's cell type (Lymphoid tissue) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species and clonality, and GZMA knockout tissue if available (standard IHC specificity controls). For chromogenic tonsil IHC, block endogenous peroxidase and check for endogenous biotin if using avidin–biotin detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the PA1588 tonsil IHC(P) caption does not report a fixative (PA1588 IHC(P) caption). Antigen-retrieval dependency is unreported, so assess retrieval conditions with the positive and negative controls (standard IHC practice). The supplied evidence does not establish whether frozen sections or IF are easier; endogenous peroxidase in lymphoid tissue can produce background in tonsil chromogenic IHC (standard IHC practice).
HPA tissue IHC evidence for GZMA
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Low consistency between antibody staining and RNA expression data. Secreted protein, tissue location of RNA and protein is expected to differ.). 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 → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Medium | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | 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 → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced GZMA IHC Tips
Use compartment, cell identity, and section controls together when troubleshooting GZMA staining in chromogenic paraffin-section IHC.
How should I adjust retrieval when GZMA staining is weak?
Start with citrate buffer at pH 6.0 for heat-induced retrieval at 95–98 °C for 20 min (page retrieval setting). Compare adjacent sections processed with and without retrieval, keeping the catalog antibody and chromogenic detection conditions identical so that a change in signal has a clear cause (standard IHC practice). If staining remains weak, test a separate section with EDTA at pH 9.0 as a fallback, and check whether background rises alongside the signal (standard IHC practice). Evaluate punctate cytoplasmic staining in immune cells, while remembering that GZMA is a granule protein with a secreted annotation and no transmembrane segment (UniProt P12544 localization and topology).
Could fixation explain weak staining in the tonsil section?
Target-specific fixation sensitivity is unknown: the PA1588 tissue caption identifies human tonsil IHC(P) but does not state its fixative (PA1588 tissue-IHC caption). Record the fixative, fixation duration, and section age for each specimen before comparing signal across runs; those variables can change antigen accessibility in paraffin-section IHC (standard IHC practice). When troubleshooting, compare similarly processed sections and change one preparation variable at a time, using an appropriately stained control section in every run (standard IHC practice). Do not treat the reported tonsil image as evidence that GZMA staining tolerates a particular fixation duration or chemistry (PA1588 tissue-IHC caption).
Where should convincing GZMA chromogenic staining appear?
Expect predominantly cytoplasmic, potentially punctate staining in suitable immune cells because GZMA is stored in cytoplasmic granules of cytotoxic T cells and NK cells (UniProt P12544 localization and function). Inspect the same cells at high magnification before scoring diffuse extracellular deposits; the protein also has a secreted annotation, so extracellular signal alone does not identify its cellular source (UniProt P12544 localization; standard IHC interpretation). Use a nuclear counterstain to distinguish cytoplasm from overlapping cells and chromogen precipitate, especially in densely populated lymphoid areas (standard IHC practice). Treat a strong, uniform nuclear pattern as discordant with the annotated granule localization and investigate controls before calling it positive (UniProt P12544 localization).
Can processing or isoforms change what this antibody detects?
GZMA has alpha and beta isoforms, a signal peptide at residues 1–26, a propeptide at 27–28, and a mature chain at 29–262 (UniProt P12544 isoforms and processing). The supplied PA1588 caption does not locate its epitope or establish whether staining distinguishes those isoforms or processing states (PA1588 tissue-IHC caption). Check the catalog antibody’s stated immunogen or epitope, if available, before interpreting an absent stain as absence of every GZMA form (standard IHC interpretation). Because glycosylation is annotated at residue 170, evaluate retrieval changes empirically rather than assuming that this modification explains a staining difference (UniProt P12544 glycosylation; standard IHC practice).
How would I follow up the IHC pattern with multiplex IF?
Use IF as a separate validation experiment and pair GZMA with a marker identifying the expected cytotoxic T-cell or NK-cell population (UniProt P12544 function; standard IF practice). Choose fluorophores after inspecting an unstained section, placing the weaker signal in a channel with low tissue autofluorescence and checking each channel with single-stain controls (standard IF practice). For an intracellular granule epitope, include a carefully titrated permeabilisation step after fixation and compare it with a no-permeabilisation control; GZMA has no transmembrane segment, but its catalog antibody’s epitope is unspecified (UniProt P12544 localization and topology; PA1588 tissue-IHC caption). Score cellular colocalisation from optical images, avoiding conclusions based solely on overlapping fluorescence in crowded tissue (standard IF practice).
What should I check when DAB stains broadly?
First examine a no-primary control and the section edges to separate antibody-dependent signal from chromogen deposition or an edge effect (standard IHC practice). Apply a peroxidase block before DAB detection, then check whether residual staining follows endogenous enzyme-rich areas rather than discrete cells (standard chromogenic IHC practice). Review blocking, washes, and the catalog antibody concentration as controlled variables, changing only one at a time and avoiding an unverified GZMA-specific dilution (standard IHC practice). Compare the resulting pattern with the expected cytoplasmic granule localization; widespread nuclear or uniform stromal color warrants investigation before it is scored as GZMA (UniProt P12544 localization; standard IHC interpretation).
How should I quantify GZMA-positive cells across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue region and count cells with convincing cytoplasmic signal, applying the same threshold and counterstain criteria to every section (UniProt P12544 localization; standard IHC quantification). Report the percentage of positive nucleated cells and, where region area is reliable, positive-cell density per mm²; state which cell population forms the denominator (standard IHC quantification). If intensity matters, report a cytoplasmic H-score using intensity grades 0–3 and the percentage at each grade, for a possible range of 0–300 (standard IHC quantification). Normalize comparisons to the same tissue compartment and eligible-cell count, and exclude folds, necrotic areas, and section edges by a consistent rule (standard IHC quantification).
How can I distinguish genuine GZMA staining from artefact?
Give greatest weight to discrete cytoplasmic signal in morphologically credible immune cells, supported by a clean no-primary control, because GZMA is a granule protease of cytotoxic T cells and NK cells (UniProt P12544 localization and function; standard IHC practice). Question staining confined to cut edges, necrotic material, or broad deposits that do not resolve into cells (standard IHC interpretation). Recheck peroxidase blocking when the DAB pattern persists without primary antibody, and investigate a predominantly nuclear pattern as a localization mismatch (standard IHC practice; UniProt P12544 localization). HPA reports medium staining in tonsil germinal center cells but flags low consistency between staining and RNA; use that pattern as context, not proof of cell identity (HPA tissue IHC).
Boster reagents
Best GZMA / Granzyme A IHC Antibodies
PA1588 is an anti-GZMA antibody with real human tonsil paraffin-section IHC data (PA1588 image caption: human tonsil IHC(P)); no IF figure is supplied (catalog: IF image alts empty).
Real IHC data
Anti-Granzyme A/GZMA Antibody ®
Cat # PA1588
PA1588 will render with a human tonsil IHC(P) figure (PA1588 image caption: human tonsil IHC(P)). Its listed applications are IHC and WB, and its listed reactivity is Human (catalog: PA1588 applications and reactivity).
Which to pick: Choose PA1588 for human paraffin-section IHC because its own figure shows human tonsil IHC(P); the fixative is unreported (PA1588 image caption: human tonsil IHC(P)). No listed SKU has IF/ICC validation or nonhuman reactivity (catalog: PA1588 applications IHC/WB; reactivity Human). Clonality is unreported, so it cannot guide selection.
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P12544 (GRAA_HUMAN, Granzyme A).
- Human Protein Atlas. GZMA tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. GZMA subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. GZMA antibody validation summary (2 antibodies).
- Identification of Key Genes for Pyroptosis-Induced Salivary Gland Inflammation in Sjogren's Syndrome Based on Microarray Data and Immunohistochemistry Analysis. Journal of inflammation research 2023 — PMC10710186.
- Proteolysis of tau by granzyme A in tauopathies generates fragments that are aggregation prone. The Biochemical journal 2024 — PMC11555691.
- A Novel Pyroptosis-Related Gene Signature for Predicting the Prognosis and the Associated Immune Infiltration in Colon Adenocarcinoma. Frontiers in oncology 2022 — PMC9330598.
- RNA-Seq analysis of chikungunya virus infection and identification of granzyme A as a major promoter of arthritic inflammation. PLoS pathogens 2017 — PMC5312928.
- PubMed PMID:3257574 — UniProt-cited evidence.
- PubMed PMID:15372022 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for GZMA (Granzyme A). Expected localisation Cytoplasmic staining in tissue (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: PA1588 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Lymphoid tissue); 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 PA1588); 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-GZMA, 0.5-1μg/ml (datasheet PA1588); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PA1588); Caveat: Secreted GZMA may stain away from its RNA source (HPA tissue IHC); Isoform / epitope: 2 isoforms; check epitope against mature chain 29–262 (UniProt); Regulation: Cytotoxic T/NK cell content affects signal (UniProt). Evidence: UniProt P12544, HPA tissue IHC · Enhanced, HPA ICC-IF · Secreted, Datasheet PA1588, PMC10710186, PMC11555691. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/GZMA/immunohistochemistry-ihc-guide.