This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
GZMB / Granzyme B · IHC design guide
Design Immunohistochemistry for GZMB
Plan paraffin-section GZMB IHC around cytoplasmic staining in subsets of immune cells (HPA tissue IHC). Use bone marrow hematopoietic cells or splenic red-pulp cells as positive tissue references, and account for secretion when interpreting the pattern (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
GZMB 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 | Selective cytoplasmic staining in immune-cell subsets (HPA tissue IHC) | |
| Staining pattern | Immune-cell subsets show cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Bone marrow+1 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep formalin fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Secretion may separate protein staining from RNA location (HPA tissue IHC) | |
| Regulation | Expression regulation is not annotated (UniProt) | |
| Isoform / epitope | No isoforms; epitopes in cleaved aa 1–20 may be lost (UniProt) |
Section 1
Recommended GZMB IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet) is paired with two published GZMB IHC protocols (PMC12816328; PMC12146097).
| Sample | Paraffin-embedded human T cell lymphoma tissue; fixative not specified (datasheet M00353-1) |
| Fixation | Image fixative and duration unreported (datasheet M00353-1); 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 monoclonal (clone AAHC-7) anti-GZMB, 1:50-1:200 (datasheet M00353-1) |
| 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 | GZMB-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: Medium). HPA tissue profile: Selective cytoplasmic expression in subsets 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 rule); EDTA pH 9.0 is a published alternative (PMC12146097).
Section 2
What Is the Expected GZMB Staining Pattern?
In paraffin-section IHC, expect selective cytoplasmic GZMB staining in subsets of immune cells, consistent with its cytolytic-granule location (HPA: selective cytoplasmic expression; UniProt P10144: cytolytic granule). Hematopoietic cells in bone marrow and cells in splenic red pulp show medium staining (HPA: tissue IHC). The tissue profile has Enhanced reliability; GZMB has no transmembrane segment and is also secreted, so extracellular signal needs cautious interpretation (HPA: Enhanced; UniProt P10144: topology and location).
| Discrete cytoplasmic staining in scattered immune cells, with stronger representation in the expected positive areas. | This fits the selective tissue pattern (HPA: selective cytoplasmic expression). Granule-associated staining is plausible because GZMB resides in cytolytic granules of cytotoxic T cells and NK cells (UniProt P10144: function and location). HPA does not establish the identity of every stained cell on a section. |
| Predominantly nuclear, uniform membranous, or widespread staining across unrelated cell types. | Reassess specificity and detection artefact: HPA reports selective cytoplasmic tissue staining, while UniProt reports no transmembrane segment (HPA: tissue IHC; UniProt P10144: topology). Secreted GZMB can complicate extracellular interpretation (UniProt P10144: secreted); compartment alone does not prove whether extracellular signal is specific. |
| Strong staining of adipocytes, respiratory epithelium, or glandular cells instead of a selective immune-cell subset. | Check cross-reactivity and endogenous detection activity with controls. These cell classes are reported as not detected in the listed adipose tissue, bronchus, adrenal gland, and cervix samples (HPA: tissue IHC). Compare the exact cell class and tissue; a negative HPA observation is not a universal exclusion. |
| Diffuse chromogen over most of the section, including areas without identifiable positive cells. | Treat the distribution as background until controls resolve it. A broad haze does not resemble the selective cytoplasmic profile (HPA: tissue IHC). As general IHC practice, assess primary-antibody omission, endogenous enzyme activity, blocking, washes, and chromogen development. |
| No staining in bone-marrow hematopoietic cells or splenic red-pulp cells. | Investigate the assay before calling the study tissue negative: both are reported at medium staining level (HPA: tissue IHC). Their positive-cell subsets still require inspection; absence from every cell is not expected from a selective immune-cell pattern (HPA: tissue IHC). |
💡Expected GZMB appearanceCall an IHC result positive when identifiable immune-cell subsets show selective, discrete cytoplasmic staining, with medium-level signal possible in bone-marrow hematopoietic cells or splenic red-pulp cells; broad nuclear, membranous, or tissue-wide chromogen is suspect (HPA: tissue IHC; UniProt P10144: cytolytic granule and topology).
| Cell population and control tissue (HPA: tissue IHC) | Bone-marrow hematopoietic cells and splenic red-pulp cells are reported at medium level; appendix lymphoid tissue, lymph-node germinal-center cells, and tonsil germinal-center cells are low (HPA: tissue IHC). Choose controls by the reported cell population, then look for selective staining rather than demanding uniform positivity. |
| Granule location and secretion (UniProt P10144: location) | GZMB is associated with cytolytic granules and is secreted (UniProt P10144: location). Cytoplasmic immune-cell staining aligns with HPA tissue observations (HPA: tissue IHC). Extracellular color alone is harder to assign to a producing cell and warrants comparison with controls. |
| Protein processing and epitope information (UniProt P10144: processing) | The precursor has a signal peptide at residues 1–18 and propeptide at 19–20; the annotated chain spans 21–247 (UniProt P10144: processing). Glycosylation sites are annotated at 71 and 104 (UniProt P10144: glycosylation). Without the antibody epitope, these facts cannot predict retrieval choice or staining strength. |
| Antibody validation (HPA: antibody validation) | HPA003418 and CAB000376 each have Enhanced IHC status (HPA: antibody validation). This supports the reported tissue pattern, but does not validate an unspecified catalog antibody, dilution, retrieval condition, or every positive cell in a new specimen. |
| IF/ICC: should its signal define the paraffin-section IHC pattern? | No. HPA reports an approved calyx location in sperm by ICC-IF, while its tissue IHC profile is selective cytoplasmic staining in immune-cell subsets (HPA: subcellular ICC-IF; HPA: tissue IHC). Read the sperm observation in its own sample and assay context. |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive-control section is blank. | Positive cells may be missed, or a general IHC step may have failed; the supplied sources do not establish a GZMB-specific fixation or retrieval effect. | Inspect bone-marrow hematopoietic cells or splenic red pulp reported at medium level (HPA: tissue IHC). As general IHC practice, verify control staining, retrieval, primary incubation, and detection steps before interpreting study negatives. |
| All cells look positive. | Uniform staining conflicts with selective cytoplasmic expression (HPA: tissue IHC); background or overdevelopment is possible under general IHC practice. | Compare a primary-antibody omission control, shorten chromogen development if excessive, and review washes and blocking as general IHC practice. Score only cell-associated staining that remains interpretable. |
| Nuclei or cell borders dominate the signal. | That pattern conflicts with HPA tissue cytoplasmic staining and UniProt granule location; GZMB has no transmembrane segment (HPA: tissue IHC; UniProt P10144: location and topology). | Check the counterstain and detection controls, then compare compartment and cell identity against a known-positive section. Treat the discordant pattern as unconfirmed until controls support it. |
| Unexpected epithelial or adipocyte staining appears. | Cross-reactivity or endogenous detection activity may explain the signal. HPA reports no detection in respiratory epithelial cells of bronchus and adipocytes of adipose tissue (HPA: tissue IHC). | Compare those specific cell types with the immune-cell pattern, run a primary-antibody omission control, and address endogenous enzyme activity as general chromogenic IHC practice. |
| Only extracellular or diffuse signal is visible. | Secreted GZMB is annotated (UniProt P10144: location), but diffuse chromogen can also be nonspecific under general IHC practice. HPA tissue IHC describes selective cytoplasmic cellular staining (HPA: tissue IHC). | Record extracellular signal separately from positive cells. Use detection controls and a positive tissue section to decide whether the cellular pattern is reproducible; avoid assigning the diffuse signal to a particular cell. |
| A low-level lymphoid sample is negative while the positive control works. | The expected signal may be sparse: HPA lists low staining in appendix lymphoid tissue and lymph-node or tonsil germinal-center cells (HPA: tissue IHC). | Review the relevant cell populations and section area before scoring. Keep the medium-level bone-marrow or spleen control in the run (HPA: tissue IHC), and report a negative study section without claiming GZMB is absent from every immune cell. |
Experiment evidence
IHC & IF images showing GZMB staining
M00353-1 · IHCImmunohistochemical analysis of paraffin-embedded human T cell lymphoma, using Granzyme B Antibody.
M00353-3 · IHCHuman spleen was stained with anti-Granzyme B rabbit antibody
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000100453-GZMB/tissue/cerebral+cortex#imid_1254188
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000100453-GZMB/tissue/colon#imid_1254114
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000100453-GZMB/tissue/liver#imid_1254107
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000100453-GZMB/tissue/kidney#imid_1254124
HPA · ICC-IF SpermHuman Protein Atlas immunofluorescence, Sperm, antibody HPA003418; green = GZMB, blue = nucleus, red = microtubules.
HPA · ICC-IF SpermHuman Protein Atlas immunofluorescence, Sperm, antibody HPA003418; green = GZMB, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for GZMB IHC & IF
🧪Run spleen first and confirm staining in cells of the red pulp (HPA: spleen, cells in red pulp, Medium). Run adipose tissue as a negative comparator (HPA: adipocytes, Not detected); on the spleen slide, cells outside the stained red-pulp population should show only counterstain or background, and should be treated as internal negatives only after their identity is verified (HPA: spleen, cells in red pulp, Medium).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show GZMB in Sperm, with annotated localisation: Calyx (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a host-species-matched irrelevant IgG control matched to the monoclonal subclass when applicable, and GZMB-knockout tissue as a biological negative (standard IHC control practice). For spleen, quench endogenous peroxidase and inspect red-pulp background; block endogenous biotin if using avidin-biotin detection (HPA: spleen, cells in red pulp, Medium; standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window and antigen-retrieval dependency are unreported in the supplied evidence; the selected paraffin-section caption does not state a fixative and does not establish fixation (selected-SKU M00353-1 caption: fixative not stated). No matched frozen-section or IF/ICC protocol is supplied, so neither can be judged easier than IHC-P from this evidence (supplied application evidence: IHC-P caption only). Blood-rich spleen red pulp can complicate chromogenic scoring through endogenous peroxidase and background staining (HPA: spleen, cells in red pulp, Medium; standard IHC practice). The selected M00353-1 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image M00353-1).
HPA tissue IHC evidence for GZMB
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data. At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | 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 → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced GZMB IHC Tips
These questions address GZMB staining in paraffin sections with chromogenic detection, with one entry for the separate IF/ICC workflow.
How should I retrieve GZMB in paraffin sections when staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 minutes (page retrieval rule). If staining is weak, compare a fresh section using EDTA at pH 9 as a fallback, while holding antibody concentration and detection conditions constant (standard IHC practice). Run a positive control containing immune cells alongside both retrieval conditions; GZMB staining should be selective and cytoplasmic in subsets of immune cells (HPA tissue IHC). Judge improvement by signal in those cells and by background in neighboring cells, since stronger overall color alone does not establish better antigen retrieval (standard IHC practice).
Can I infer the best fixative for GZMB from the available tissue images?
The selected image shows paraffin-embedded human T cell lymphoma, but its caption does not state a fixative (caption: M00353-1). Consequently, target-specific GZMB sensitivity to fixation is unknown; neither the HPA staining pattern nor GZMB processing and glycosylation establishes a preferred fixative (HPA tissue IHC; UniProt P10144 processing). For specimens with known processing, record fixative, fixation duration, section thickness, and retrieval conditions before comparing staining intensity across cases (standard IHC practice). If a processing effect is suspected, compare matched material using citrate pH 6.0 retrieval for 20 minutes and the same antibody and detection run (page retrieval rule; standard IHC practice).
What staining compartment should count as GZMB positive in tissue?
Look for selective cytoplasmic staining in subsets of immune cells, the reported tissue pattern for GZMB (HPA tissue IHC). GZMB occupies cytolytic granules and can be secreted or delivered into target cells by perforin, so interpret staining against cell morphology and location rather than requiring every positive cell to have identical granule contrast (UniProt P10144 localisation). Its sequence has no transmembrane segment, so continuous membrane-only staining needs independent validation before it is scored as GZMB (UniProt P10144 topology; standard IHC practice). Review a nuclear counterstain and adjacent tissue at the same magnification, and document whether signal is cellular, extracellular, or confined to damaged areas (standard IHC practice).
Could GZMB processing or glycosylation explain inconsistent antibody staining?
GZMB has no annotated isoforms in the supplied record, but its precursor loses a signal peptide at residues 1–18 and a propeptide at 19–20 (UniProt P10144 processing and isoforms). The annotated mature chain spans 21–247, with glycosylation sites at residues 71 and 104 (UniProt P10144 processing and glycosylation). Without an epitope map for the catalog antibody, do not assign weak paraffin-section staining to either cleavage or glycosylation (catalog evidence supplied; standard IHC practice). First compare matched sections under citrate pH 6.0 retrieval for 20 minutes, then investigate epitope accessibility only if controls support a reproducible discrepancy (page retrieval rule; standard IHC practice).
How can I check GZMB localisation by multiplex IF/ICC?
Pair GZMB with a marker that identifies the expected cytotoxic immune-cell population, and inspect both channels in individual cells before interpreting overlap (UniProt P10144 function; standard IF practice). Choose spectrally separated fluorophores and place the weaker signal in a channel with low tissue autofluorescence, checking unstained and single-color controls under the same imaging settings (standard IF practice). Because GZMB is found in cytolytic granules rather than across a transmembrane segment, use a validated permeabilisation step to expose an intracellular epitope, then compare with an unpermeabilised control if epitope access is uncertain (UniProt P10144 localisation and topology; standard IF practice). Optimise IF/ICC fixation and permeabilisation on IF/ICC material; the paraffin IHC caption supplies no IF/ICC fixation evidence (caption: M00353-1).
How do I distinguish GZMB signal from chromogenic background?
Inspect a no-primary control and an expected negative cell population on the same run before accepting diffuse brown staining as GZMB (standard IHC practice). Block endogenous peroxidase before a peroxidase and DAB detection workflow, and check whether pigment or tissue deposits remain visible without primary antibody (standard IHC practice). HPA reports GZMB as not detected in adipocytes from adipose tissue, whereas its tissue pattern is selective cytoplasmic staining in immune-cell subsets (HPA tissue IHC). If background rises after citrate pH 6.0 retrieval for 20 minutes, compare sections processed together and adjust blocking, washing, or detection conditions one variable at a time (page retrieval rule; standard IHC practice).
What should I score when comparing GZMB staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue area before scoring, because GZMB expression is selective among immune cells rather than uniform across tissue (HPA tissue IHC). Report the percentage of positive cells within the defined population, or positive-cell density per mm² of evaluable tissue; retain the denominator and exclusion rules with each result (standard IHC practice). If intensity matters, use a prespecified 0–3 intensity scale and calculate an H-score from intensity-weighted percentages, with a possible range of 0–300 (standard IHC practice). Exclude folds, edges, and necrotic areas consistently, and compare only sections with documented processing and matched staining settings (standard IHC practice).
Which apparent GZMB positives need closer review before reporting?
A convincing result is selective cytoplasmic signal in morphologically appropriate immune cells, supported by the reported HPA tissue pattern and GZMB localisation in cytolytic granules (HPA tissue IHC; UniProt P10144 localisation). Treat uniform nuclear or membrane-only color, staining restricted to section edges or necrosis, and widespread signal in morphologically unrelated cells as findings that need controls before interpretation (UniProt P10144 topology; standard IHC practice). Check a no-primary slide for endogenous enzyme signal or pigment, and review the counterstain to assign each positive focus to an intact cell (standard IHC practice). Because secreted GZMB can leave its source cell, extracellular color alone should not be counted as a GZMB-positive immune cell (UniProt P10144 localisation; standard IHC practice).
Boster reagents
Best GZMB / Granzyme B IHC Antibodies
Two human-reactive anti-GZMB antibodies have IHC images from paraffin-embedded human T cell lymphoma and human spleen (catalog image captions). Both list IF, but neither has an IF figure (catalog applications and images).
Real IHC data
Anti-Granzyme B GZMB Monoclonal Antibody
Cat # M00353-1
Real IHC data
Anti-Granzyme B Rabbit Monoclonal Antibody
Cat # M00353-3
M00353-1 lists Human IHC, IF and ICC, with an IHC image captioned as paraffin-embedded human T cell lymphoma (catalog applications, reactivity and image caption). M00353-3 lists Human IHC and IF, with an IHC image captioned as human spleen; its tissue processing is unreported (catalog applications, reactivity and image caption).
Which to pick: For paraffin-section tissue IHC, choose M00353-1: its own image caption specifies paraffin-embedded human T cell lymphoma, but does not report the fixative (catalog image caption). For IF/ICC, choose M00353-1 if ICC is required; both M00353-1 and M00353-3 list IF, with 1:50–1:200 and 1:200–1:1000 catalog dilutions respectively (catalog applications and dilutions). For Human/Mouse IHC, consider M00353-4 or PA1738; PA1738 also lists Rat reactivity, but its stated IHC-P species are Human and Mouse (catalog reactivity and application data).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P10144 (GRAB_HUMAN, Granzyme B).
- Human Protein Atlas. GZMB tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. GZMB subcellular location (ICC-IF): Localized to the calyx..
- Human Protein Atlas. GZMB antibody validation summary (2 antibodies).
- Unique Spatial Immune Profiling in Pancreatic Ductal Adenocarcinoma with Enrichment of Exhausted and Senescent T Cells and Diffused CD47-SIRPα Expression. Cancers 2020 — PMC7408661.
- Integrative analysis of bulk and single-cell transcriptomics identifies factors related to immunosuppressive microenvironment to predict unfavorable prognosis in inflammatory breast cancer. Frontiers in immunology 2025 — PMC12816328.
- Development and Validation of an Anoikis-Related Gene Signature for Prognostic Prediction in Cervical Cancer. International journal of general medicine 2025 — PMC12146097.
- The pro-apoptotic GZMB and anti-apoptotic HAX-1 and DAD-1 genes and gene proteins in human right auricular tissue affected by coronary heart disease and aortic valve stenosis: a pilot study. Journal of molecular histology 2026 — PMC13264547.
- PubMed PMID:2953813 — UniProt-cited evidence.
- PubMed PMID:3258865 — UniProt-cited evidence.
- PubMed PMID:3261871 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for GZMB (Granzyme B). Expected localisation Selective cytoplasmic staining in immune-cell subsets (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: M00353-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human T cell lymphoma tissue; fixative not specified (datasheet M00353-1); 2. Fixation: Image fixative and duration unreported (datasheet M00353-1); 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 monoclonal (clone AAHC-7) anti-GZMB, 1:50-1:200 (datasheet M00353-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep formalin fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Secretion may separate protein staining from RNA location (HPA tissue IHC); Isoform / epitope: No isoforms; epitopes in cleaved aa 1–20 may be lost (UniProt); Regulation: Expression regulation is not annotated (UniProt). Evidence: UniProt P10144, HPA tissue IHC · Enhanced, HPA ICC-IF · calyx., Datasheet M00353-1, PMC12816328, PMC12146097. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/GZMB/immunohistochemistry-ihc-guide.