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GZMB / Granzyme B · IHC design guide
Design Immunohistochemistry for GZMB
Plan paraffin section IHC for GZMB using selective cytoplasmic staining in immune cell subsets as the expected tissue pattern (HPA tissue IHC). Compare antibody dilution options and interpret staining with its secreted form in mind (datasheet; 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 | Cytoplasm in immune cell subsets (HPA tissue IHC); cytolytic granules and secreted (UniProt) | |
| Staining pattern | 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) | |
| 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 fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Secreted protein may differ in location from its RNA (HPA tissue IHC) | |
| Regulation | Expression regulation not annotated (UniProt) | |
| Isoform / epitope | No isoforms; mature chain starts at residue 21 (UniProt) |
Section 1
Recommended GZMB IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with four published GZMB IHC protocols covering pancreatic, breast, cervical, and right auricular tissue (PMC7408661; PMC12816328; PMC12146097; PMC13264547).
| 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 was used for cervical tissue microarrays (PMC12146097).
Section 2
What Is the Expected GZMB Staining Pattern?
GZMB is stored in cytolytic granules and has no transmembrane segment (UniProt P10144). In paraffin IHC, expect selective cytoplasmic staining in subsets of immune cells (HPA tissue IHC: Enhanced). Cytotoxic T cells and NK cells contain GZMB (UniProt P10144 function).
| Cytoplasmic stain in scattered immune cells. | Expected selective pattern (HPA tissue IHC: Enhanced). |
| Predominantly nuclear or membrane stain. | Discordant with cytoplasmic IHC (HPA tissue IHC) and granule location (UniProt P10144). |
| Widespread adipocyte staining. | Suspect nonspecific signal; adipocytes are not detected (HPA tissue IHC). |
| Uniform haze across the section. | Background obscures the selective immune-cell pattern (HPA tissue IHC). |
| No stain in marrow hematopoietic cells. | Unexpected; these cells show medium staining (HPA tissue IHC). |
💡Expected GZMB appearanceCall positive for selective cytoplasmic staining in immune cells, including medium staining in marrow hematopoietic cells; widespread adipocyte staining is suspect (HPA tissue IHC).
| Positive tissue reference | Marrow hematopoietic and spleen red-pulp cells stain medium (HPA tissue IHC). |
| Lower-signal sites | Appendix lymphoid and lymph-node or tonsil germinal-center cells stain low (HPA tissue IHC). |
| Protein location | Cytolytic granule and secreted; no transmembrane segment (UniProt P10144). |
| Antibody validation | HPA003418 and CAB000376 have Enhanced IHC validation (HPA antibodies). |
| IF/ICC: expected location? | Calyx in sperm (HPA ICC-IF: approved); see the separate IF/ICC guide. |
| Situation | Likely cause | Next action |
|---|---|---|
| No marrow signal | Marrow hematopoietic cells stain medium (HPA tissue IHC). | Check retrieval, primary dilution and detection with a positive control (standard IHC practice). |
| Predominantly nuclear stain | Discordant with cytoplasmic pattern (HPA tissue IHC). | Check a no-primary control and detection conditions (standard IHC practice). |
| Adipocytes stain | Adipocytes are not detected (HPA tissue IHC); nonspecific signal is possible. | Compare with a negative control and the immune-cell pattern (standard IHC practice). |
| Diffuse chromogen haze | If using HRP, endogenous peroxidase may contribute (standard IHC practice). | Use a peroxidase block and no-primary control (standard IHC practice). |
| Weak lymph-node signal | Germinal-center cells show low staining (HPA tissue IHC). | Compare with marrow hematopoietic cells: medium (HPA tissue IHC). |
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: cells in the red pulp should stain at a medium level (HPA: Spleen, cells in red pulp, Medium). Use adipose tissue as the negative, checking that adipocytes lack staining (HPA: Adipose tissue, adipocytes, Not detected); on the spleen slide, cells outside the stained red-pulp population should lack specific signal (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 nonimmune isotype control matched to the primary antibody’s host species, immunoglobulin class and clonality, and GZMB-knockout material or a validated peptide-blocking control if available (standard IHC/IF practice). For spleen red pulp, block endogenous peroxidase in chromogenic IHC and assess autofluorescence if using IF (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window and the effect of antigen retrieval are unreported in the supplied evidence; optimize retrieval for the catalog antibody on paraffin sections (standard IHC practice). The selected M00353-1 paraffin-section caption does not report a fixative (selected tissue-IHC caption: fixative not stated). The supplied evidence does not establish whether frozen sections or IF are easier; red-pulp endogenous peroxidase can complicate chromogenic interpretation (standard IHC practice).
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
Troubleshoot GZMB staining in paraffin sections by checking retrieval, cellular localisation, controls and how positive immune cells are counted.
What should I change when GZMB staining is weak after antigen retrieval?
Start with citrate pH 6.0 heat-induced retrieval at 95–98 °C for 20 min (page retrieval rule). Keep the antibody dilution and detection conditions constant while checking whether the section reached the stated temperature throughout the run (standard IHC practice). Compare treated sections with a retrieval-omitted section and a positive-control section in the same run (standard IHC practice). Score staining in immune-cell cytoplasm, because selective cytoplasmic staining is the tissue pattern reported for GZMB (HPA tissue IHC). If signal remains weak, optimise heating or cooling within the citrate workflow before comparing a different retrieval condition (standard IHC practice).
Can fixation explain weak GZMB staining in these paraffin sections?
Target-specific fixation sensitivity is unknown from the supplied evidence; the selected antibody caption identifies paraffin-embedded human T cell lymphoma but does not state its fixative (catalog antibody caption: M00353-1). Record fixative, fixation duration and tissue thickness for each specimen before comparing staining intensity across runs (standard IHC practice). Use matched processing and the same citrate pH 6.0 retrieval at 95–98 °C for 20 min when testing whether preparation differences track the weak signal (page retrieval rule). Inspect morphology alongside the chromogen to identify poorly preserved regions (standard IHC practice). Do not assign a GZMB-specific fixation effect without a controlled comparison of matched sections (supplied fixation evidence).
Where should a convincing GZMB signal appear in tissue IHC?
Look first for selective cytoplasmic staining in subsets of immune cells (HPA tissue IHC). Granzyme B is associated with cytolytic granules in cytotoxic T cells and NK cells and can be delivered into a target cell by perforin, so interpret any target-cell signal in its cellular context (UniProt P10144 function and subcellular annotation). The protein has no transmembrane segment, so a uniform surface outline alone is a poor match to its annotated topology (UniProt P10144 topology). Compare staining with a nuclear counterstain and cell morphology on the same section (standard IHC practice). HPA also reports a calyx location in sperm ICC/IF images; that observation does not define the expected pattern in these tissue sections (HPA subcellular).
How can GZMB processing affect the epitope seen by IHC?
GZMB has no annotated isoforms in the supplied record, but its precursor contains a signal peptide at residues 1–18, a propeptide at 19–20 and a mature chain at 21–247 (UniProt P10144 processing and isoforms). Two glycosylation sites are annotated at residues 71 and 104 (UniProt P10144 glycosylation). The catalog caption does not identify the antibody epitope, so these annotations cannot establish which molecular form it detects (catalog antibody caption: M00353-1). If staining differs between specimens, compare sections under identical retrieval and detection conditions before attributing the difference to processing (standard IHC practice). Ask for epitope information before interpreting an apparent compartment-specific loss of signal (standard IHC practice).
How should IF corroborate the GZMB pattern seen by chromogenic IHC?
Use IF as a separate corroborating experiment, comparing its cellular pattern with the cytoplasmic immune-cell pattern expected in tissue IHC (HPA tissue IHC). Multiplex GZMB with a cytotoxic T-cell or NK-cell marker, such as CD8 or CD56, while retaining single-stain controls to assess channel bleed-through (UniProt P10144 function; standard IF practice). Choose a fluorophore away from the specimen's strongest autofluorescence and check an unstained section in each imaging channel (standard IF practice). Because GZMB is granule-associated and lacks a transmembrane segment, permeabilise fixed cells for intracellular epitope access, then optimise detergent strength against morphology (UniProt P10144 subcellular annotation and topology; standard IF practice). The paraffin IHC caption supplies no IF fixation condition (catalog antibody caption: M00353-1).
How do I reduce diffuse or widespread brown staining?
First compare the specimen with a primary-antibody-omitted control to separate detection background from antibody-associated staining (standard IHC practice). Include an appropriate peroxidase block before chromogenic detection, and inspect a control for endogenous enzyme activity, especially when evaluating blood-rich tissue (standard IHC practice). Titrate the primary antibody and adjust wash or blocking conditions one variable at a time while preserving the same retrieval settings (standard IHC practice; page retrieval rule). Prioritise discrete cytoplasmic immune-cell staining over diffuse colour, because the reported GZMB tissue profile is selective in immune-cell subsets (HPA tissue IHC). Exclude folds, edges and damaged areas from the comparison before changing the staining protocol (standard IHC practice).
What should I count when quantifying GZMB-positive tissue? ⚠ ANSWER MARKED FOR VERIFICATION
Define positivity as cytoplasmic staining in morphologically identifiable cells before measuring the section (HPA tissue IHC; standard IHC practice). Report the percentage of positive cells within a specified viable cell population, or count positive cells per mm² of viable tissue when cell density varies (standard IHC practice). An H-score can combine intensity categories 0–3 with the percentage of cells in each category, giving a 0–300 range (standard IHC practice). Keep the compartment, intensity threshold and sampled area consistent across specimens (standard IHC practice). State the denominator explicitly, since GZMB expression is selective among immune-cell subsets rather than uniformly distributed through tissue (HPA tissue IHC).
How can I distinguish true GZMB positivity from staining artefact?
A plausible positive result is discrete cytoplasmic staining in an immune-cell subset, consistent with the reported tissue pattern and granule-associated function (HPA tissue IHC; UniProt P10144 function). Check the counterstain and morphology before assigning brown signal to a cell, especially at crowded boundaries (standard IHC practice). Treat isolated nuclear staining or a uniform cell-surface outline cautiously because neither matches the reported cytoplasmic pattern or the absence of a transmembrane segment (HPA tissue IHC; UniProt P10144 topology). Compare section edges and necrotic areas with intact interior tissue, and use a primary-antibody-omitted control to assess endogenous enzyme or detection signal (standard IHC practice). Interpret possible target-cell staining in context, since perforin can deliver GZMB into target cells (UniProt P10144 subcellular annotation).
Boster reagents
Best GZMB / Granzyme B IHC Antibodies
Catalog figures show IHC in human paraffin-embedded T cell lymphoma and human spleen (image alts); the catalog also lists human IF, human/mouse IHC, and rat reactivity (catalog applications/reactivity).
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 has an IHC figure from paraffin-embedded human T cell lymphoma and is listed for human IHC, IF, and ICC (image alt; catalog applications/reactivity). M00353-3 has an IHC figure from human spleen and is listed for human IHC and IF (image alt; catalog applications/reactivity).
Which to pick: For tissue IHC, choose M00353-1 when the sample is a paraffin section of human T cell lymphoma (image alt), or M00353-3 for human spleen (image alt); the fixative is unreported for both, and processing is unreported for the spleen figure (image alts). For IF/ICC, M00353-1 lists both applications, while M00353-3 lists IF only (catalog applications). For human or mouse IHC, M00353-4 is a rabbit monoclonal option (catalog host/clone/applications/reactivity); PA1738 also lists human and mouse IHC, while its rat reactivity listing does not establish rat IHC validation (catalog dilution_raw/applications/reactivity).
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 Cytoplasm in immune cell subsets (HPA tissue IHC); cytolytic granules and secreted (UniProt). 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 fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Secreted protein may differ in location from its RNA (HPA tissue IHC); Isoform / epitope: No isoforms; mature chain starts at residue 21 (UniProt); Regulation: Expression regulation not annotated (UniProt). Evidence: UniProt P10144, HPA tissue IHC · Enhanced, HPA ICC-IF · calyx., Datasheet M00353-1, PMC7408661, PMC12816328. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/GZMB/immunohistochemistry-ihc-guide.