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GLS2 / Glutaminase liver isoform, mitochondrial · IHC design guide
Design Immunohistochemistry for GLS2
Plan GLS2 chromogenic IHC in paraffin sections using the documented fixation and antibody conditions (datasheet A05334). Compare cytoplasmic staining with the tissue profile while accounting for its uncertain specificity (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
GLS2 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 tissue staining (HPA tissue IHC); mitochondrial protein (UniProt) | |
| Staining pattern | Cytoplasmic staining in liver, CNS and pancreas (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A05334) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+2 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Use formaldehyde-fixed paraffin sections (selected-SKU IHC image A05334); keep fixation consistent (standard IHC practice; not target-specific) | |
| Caveat | Presumed off-target binding; verify specificity (HPA tissue IHC) | |
| Regulation | Reduced in hepatocellular carcinomas (UniProt) | |
| Isoform / epitope | Two isoforms; map epitopes to mature chain 15–602 (UniProt) |
Section 1
Recommended GLS2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet A05334) is accompanied by two published GLS2 protocols for paraffin sections (PMC4480704; PMC7215180).
| Sample | Formaldehyde-fixed, paraffin-embedded human liver tissue (datasheet A05334) |
| Fixation | Image formalin-fixed; duration unreported (datasheet A05334); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6 (datasheet A05334); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% serum (datasheet A05334) |
| Primary antibody | Rabbit anti-GLS2, 0.5-2 μg/mL (datasheet A05334) |
| 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 | GLS2-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in liver, CNS and pancreas. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate retrieval at pH 6 (datasheet A05334); PMC7215180 reports 10 minutes in 10 mmol/L sodium citrate.
Section 2
What Is the Expected GLS2 Staining Pattern?
GLS2 is mitochondrial and has no transmembrane segment (UniProt Q9UI32 topology). In paraffin section IHC, expect cytoplasmic staining in liver, CNS and pancreas, with liver the strongest tissue expectation from UniProt expression (HPA: tissue IHC profile; UniProt Q9UI32 tissue specificity). Interpret that pattern cautiously: HPA rates its tissue IHC reliability Uncertain because presumed off target binding was observed and disregarded, pending external verification (HPA: tissue IHC reliability).
| Cytoplasmic staining in liver cells, with a granular appearance if resolved. | This fits the reported liver expression and mitochondrial location (HPA: tissue IHC profile; UniProt Q9UI32 location). Granularity is a plausible mitochondrial appearance in chromogenic IHC, not a validated GLS2 scoring requirement (general IHC practice). Compare signal with adjacent tissue and controls before calling it specific, given HPA's Uncertain IHC reliability (HPA: tissue IHC reliability). |
| Predominantly nuclear, cell surface or extracellular staining. | These compartments conflict with the mitochondrial assignment and lack of a transmembrane segment (UniProt Q9UI32 location and topology). Treat a compartment mismatch as possible nonspecific staining or artefact, particularly if cytoplasmic signal is absent (general IHC practice); HPA does not provide a verified alternate GLS2 location here (HPA: tissue IHC reliability). |
| Strong staining in a cell population expected to be negative on the comparison slide. | Assess the cell type, not just the tissue name: HPA reports no detection in skeletal muscle myocytes, adipocytes and smooth muscle cells, but High staining in several glandular or squamous epithelial populations (HPA: tissue IHC levels). Unexpected staining can reflect cross reactivity or endogenous detection activity; it does not establish GLS2 expression on its own (general IHC practice). |
| Uniform color over cells, stroma and empty areas. | A diffuse deposit without cell boundaries or a cytoplasmic pattern is poor evidence for mitochondrial GLS2 (UniProt Q9UI32 location; general IHC practice). Consider nonspecific antibody binding, inadequate blocking, chromogen overdevelopment or endogenous detection activity as general IHC causes; HPA has flagged presumed off target binding for its tissue IHC profile (HPA: tissue IHC reliability). |
| No visible staining in liver on an otherwise readable section. | This is discordant with high liver expression and HPA's liver profile (UniProt Q9UI32 tissue specificity; HPA: tissue IHC profile). First check whether tissue, counterstain and detection controls are interpretable; then review retrieval, antibody dilution and detection settings as general IHC variables (general IHC practice). An absent signal alone cannot distinguish assay failure from specimen variation. |
💡Expected GLS2 appearanceCall a positive when staining is chiefly cytoplasmic in the expected liver cell population, plausibly granular and stronger than background (UniProt Q9UI32 location and liver expression; HPA: tissue IHC profile); isolated nuclear, surface or diffuse stromal color is a suspect positive (UniProt Q9UI32 topology; general IHC practice), and HPA rates its IHC evidence Uncertain (HPA: tissue IHC reliability).
| Tissue and cell context | UniProt reports high liver expression, with expression in brain and pancreas, while HPA describes cytoplasmic staining in liver, CNS and pancreas (UniProt Q9UI32 tissue specificity; HPA: tissue IHC profile). HPA also lists High staining in selected glandular and squamous cells outside that summary, but its IHC reliability is Uncertain; use those entries as observations to verify, not definitive specificity controls (HPA: tissue IHC levels and reliability). |
| Intracellular location and processing | GLS2 is assigned to mitochondria, lacks a transmembrane segment and has a reported mature chain spanning residues 15–602 (UniProt Q9UI32 location, topology and processing). These annotations support a cytoplasmic mitochondrial pattern, but do not identify the antibody epitope or predict the appearance of every processed form in a section (UniProt Q9UI32 processing; general IHC practice). |
| Isoforms and epitope coverage | UniProt lists 2 isoforms, but the supplied records do not locate this antibody's epitope or establish isoform specific IHC recognition (UniProt Q9UI32 isoforms; HPA: antibody record). If staining differs among samples, do not assign the difference to an isoform without separate epitope and specificity evidence (general IHC practice). |
| Antibody evidence and controls | HPA lists rabbit polyclonal HPA038608 with IHC status Uncertain; its tissue profile notes presumed off target binding and pending external verification (HPA: antibody record and tissue IHC reliability). Interpret its pattern alongside a matched negative control and an independently supported positive tissue, while recognising that a clean control cannot by itself prove target specificity (general IHC practice). |
| IF/ICC: what pattern is established? | Mitochondrial signal is the expectation from UniProt, but HPA supplies no ICC/IF images or main subcellular location for GLS2 (UniProt Q9UI32 location; HPA: subcellular record). Treat an IF/ICC pattern as a separate question requiring its own validation; the tissue IHC observations do not establish an IF/ICC result (HPA: tissue IHC and subcellular records). |
| Situation | Likely cause | Next action |
|---|---|---|
| Liver signal is absent or very weak. | High liver expression makes this discordant, though the supplied sources give no target specific fixation or retrieval sensitivity (UniProt Q9UI32 tissue specificity; HPA: tissue IHC profile). | Check section integrity and detection controls, then compare retrieval and antibody dilution conditions as general IHC variables; record which change restores a cell restricted pattern (general IHC practice). |
| A negative comparison tissue shows strong color. | HPA reports no detection in skeletal muscle myocytes, adipocytes and smooth muscle cells; color there may reflect nonspecific binding or endogenous detection activity (HPA: tissue IHC levels; general IHC practice). | Confirm the stained cell type, inspect the negative reagent control, and adjust blocking or detection conditions only as needed; reassess liver and negative tissue together (general IHC practice). |
| Nuclei dominate the stain. | A dominant nuclear pattern does not match mitochondrial GLS2 localization (UniProt Q9UI32 location). Overstain or nonspecific binding can obscure the true compartment (general IHC practice). | Compare the no primary control and shorten detection development if background is high; seek reproducible cytoplasmic signal before assigning GLS2 positivity (general IHC practice). |
| Background hides cell boundaries. | Diffuse color can arise from nonspecific binding, incomplete blocking or excessive detection development (general IHC practice); HPA's IHC reliability is Uncertain (HPA: tissue IHC reliability). | Review negative reagent controls, blocking, washes and detection development, then judge whether signal remains concentrated in the expected cells (general IHC practice). |
| An unexpected glandular or squamous cell population stains strongly. | HPA records High staining in several such populations outside its brief liver, CNS and pancreas profile, but flags presumed off target binding (HPA: tissue IHC levels, profile and reliability). | Record the tissue and cell type precisely; require independent specificity evidence before interpreting that color as GLS2 expression (general IHC practice). |
| IF/ICC shows a diffuse or conflicting pattern. | HPA provides no GLS2 ICC/IF images or assigned main subcellular location, so its tissue IHC profile cannot validate the IF/ICC result (HPA: subcellular and tissue IHC records). | Evaluate IF/ICC with its own controls and mitochondrial reference while treating UniProt's mitochondrial assignment as the localization expectation (general IF practice; UniProt Q9UI32 location). |
Experiment evidence
IHC & IF images showing GLS2 staining
A05334 · IHCImmunohistochemistry Validation of GLS2 in Human Liver Immunohistochemical analysis of paraffin-embedded human liver tissue using anti-GLS2
A05334 · IHCImmunohistochemistry Validation of GLS2 in Mouse Liver Immunohistochemical analysis of paraffin-embedded mouse liver tissue using anti-GLS2
A05334 · IFImmunofluorescence Validation of GLS2 in Mouse Brain Immunofluorescent analysis of 4% paraformaldehyde-fixed mouse brain labeling GLS2 with
M05334 · IHCM05334 staining GLS2 in human kidney tissue sections by Immunohistochemistry (IHC-P -paraformaldehyde-fixed, paraffin-embedded sections). Ti
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000135423-GLS2/tissue/cerebral+cortex#imid_9805548
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000135423-GLS2/tissue/colon#imid_9787698
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000135423-GLS2/tissue/liver#imid_9787707
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000135423-GLS2/tissue/kidney#imid_9787692
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for GLS2 IHC & IF
🧪Run colon first: glandular cells should stain strongly (HPA: High in colon glandular cells). Use skeletal muscle myocytes as the negative tissue (HPA: Not detected in skeletal muscle myocytes); smooth muscle cells on the colon slide should provide an internal unstained comparison (HPA: Not detected in smooth muscle cells).
Positive control tissue: Adrenal gland (Glandular 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 GLS2; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and clonality; use GLS2 knockout material as a biological specificity control (standard IHC practice). Block endogenous peroxidase before chromogenic detection and inspect colon luminal material for nonspecific signal (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected SKU’s fixative field is unreported (selected-SKU fixative field: not stated). The paraffin-section caption uses heat retrieval in citrate buffer at pH 6, but does not establish that retrieval is required (selected-SKU IHC caption). Frozen sections and IF have no demonstrated ease advantage in the supplied evidence; no ICC-IF images are listed (HPA subcellular record).
HPA tissue IHC evidence for GLS2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Presumed off target binding observed and disregarded. Pending external verification.). 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
Section 3
Advanced GLS2 IHC Tips
Troubleshoot GLS2 staining in paraffin sections by checking retrieval, controls, cytoplasmic localisation and cell specific scoring before interpreting signal.
What retrieval should I use when GLS2 staining is weak in paraffin sections?
Start with heat-mediated citrate retrieval at pH 6 for GLS2 paraffin sections (datasheet A05334). The selected human liver example used that retrieval, 1 µg/ml primary antibody overnight at 4°C, and a hematoxylin counterstain (caption A05334). Compare serial sections while changing only retrieval time, keeping antibody incubation and detection constant; excessive heating can damage morphology and make staining harder to assess (standard IHC practice). Include liver and no-primary controls in each run; if signal remains weak with preserved morphology, try another retrieval buffer as a fallback and reassess background (UniProt Q9UI32: liver expression; standard IHC practice).
How can I assess whether fixation is affecting GLS2 detection?
GLS2 specific sensitivity to fixation duration or fixative composition is unknown from the supplied evidence, so establish it experimentally rather than assigning a target specific effect (supplied GLS2 evidence). Compare sections with documented fixation histories using the same pH 6 citrate retrieval and matched antibody incubation (datasheet A05334; standard IHC practice). Assess morphology, signal intensity and background together, since poor preservation or uneven fixation can complicate chromogenic interpretation (standard IHC practice). Keep a liver control in each comparison and avoid interpreting a negative study section until its processing and detection controls are satisfactory (UniProt Q9UI32: liver expression; standard IHC practice).
What staining pattern should count as plausible GLS2 localisation?
Expect GLS2 signal within the cytoplasmic compartment because the protein is mitochondrial; assess whether any punctate pattern is reproducible across cells and sections (UniProt Q9UI32: mitochondrion; standard IHC practice). A predominantly nuclear or surface restricted pattern needs investigation before it is called specific, since no transmembrane segment is annotated for GLS2 (UniProt Q9UI32: topology). The tissue atlas describes cytoplasmic expression in liver, CNS and pancreas, although its staining reliability is marked uncertain because presumed off target binding was observed (HPA tissue IHC: profile and reliability). Check localisation against a liver control, a no-primary section and intact cell morphology rather than relying on chromogen intensity alone (UniProt Q9UI32: liver expression; standard IHC practice).
Could isoforms or epitope accessibility explain discordant GLS2 staining?
GLS2 has 2 annotated isoforms, so establish which sequence the antibody recognises before treating discordant staining as a biological difference (UniProt Q9UI32: isoforms; standard IHC practice). The annotated mature chain spans residues 15–602, and several modified residues are listed, but these annotations alone do not establish that a particular epitope is masked in tissue (UniProt Q9UI32: processing and modified residues). Request or inspect the immunogen sequence, then check its presence in each isoform and its position relative to the processed N terminus (standard antibody validation practice; UniProt Q9UI32: isoforms and processing). If the epitope remains unknown, compare independent epitope reagents and matched controls before assigning a negative compartment or specimen to isoform loss (standard IHC practice).
How should I investigate GLS2 by IF alongside the chromogenic IHC result?
For a separate IF experiment, multiplex GLS2 with a marker of the expected cell population, such as a hepatocyte marker in liver, and include single stain controls (UniProt Q9UI32: liver expression; standard IF practice). Choose fluorophores after examining unstained tissue autofluorescence, favouring a spectral channel with clear separation from that background (standard IF practice). Because GLS2 is mitochondrial and has no annotated transmembrane segment, permeabilise sufficiently to access the intracellular epitope while checking that cellular structure remains intact (UniProt Q9UI32: localisation and topology; standard IF practice). Compare the distribution with matched chromogenic sections, but validate the IF antibody and fixation conditions independently rather than transferring the IHC caption as IF evidence (caption A05334: IHC; standard IF practice).
How can I separate GLS2 signal from chromogenic background?
Run a no-primary section to reveal detection reagent background, and inspect unstained or substrate only sections when pigment or endogenous colour complicates interpretation (standard IHC practice). Block endogenous peroxidase before HRP and DAB detection, then evaluate whether the chromogen remains restricted to intact cellular compartments (standard chromogenic IHC practice). The selected liver example used 10% serum for 1 hour at room temperature and an HRP secondary at 1:250; these are conditions to compare, not proof of specificity (caption A05334). Titrate primary antibody around the reported 1 µg/ml while holding retrieval and exposure constant, and reject patterns dominated by section edges or tissue damage (caption A05334; standard IHC practice).
What is a defensible way to score GLS2 IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic staining threshold before scoring, then apply the same rules to every section (UniProt Q9UI32: mitochondrial localisation; standard IHC practice). An H-score combines the percentage of cells at each intensity, giving a 0–300 scale when intensities are scored 0–3 (standard IHC scoring practice). Alternatively, report the percentage of positive cells or positive cell density per mm², with the counted tissue area and exclusion rules stated (standard image analysis practice). Normalise comparisons to the relevant viable cell population or analysed tissue area, and process controls and specimens together because staining strength can vary between runs (standard IHC practice).
When should I doubt an apparent GLS2 positive IHC result?
Treat convincing GLS2 staining as intracellular cytoplasmic signal in appropriate intact cells, with a reproducible liver control and clean no-primary control (UniProt Q9UI32: mitochondrion and liver expression; standard IHC practice). Question predominantly nuclear or membranous signal, staining confined to necrosis or section edges, and diffuse colour that persists without primary antibody (UniProt Q9UI32: localisation and topology; standard IHC practice). Investigate endogenous peroxidase when HRP and DAB colour appears in the no-primary control, using a peroxidase block and matched detection controls (standard chromogenic IHC practice). Interpret unexpected cell types cautiously: the tissue atlas reports broad staining but rates its IHC reliability uncertain owing to presumed off target binding (HPA tissue IHC: positive tissues and reliability).
Boster reagents
Best GLS2 / Glutaminase liver isoform, mitochondrial IHC Antibodies
GLS2 IHC images cover human and mouse liver, rat brain, and human kidney; an IF image covers mouse brain (catalog image captions).
Real IHC data
Anti-GLS2 Antibody
Cat # A05334
Real IHC data
Anti-GLS2 Antibody
Cat # M05334
A05334 has IHC images from human and mouse liver and rat brain, plus an IF image from mouse brain (A05334 image captions). M05334 has an IHC image from human kidney (M05334 image caption).
Which to pick: For paraffin-section IHC, choose A05334 for the pictured liver or brain samples, or M05334 for the pictured human kidney sample; both captions report formaldehyde fixation and citrate pH 6 retrieval (A05334 and M05334 IHC captions). For IF, choose A05334: its mouse-brain IF image uses paraformaldehyde-fixed tissue; ICC validation is unreported (A05334 IF caption and applications; M05334 applications). For cross-species IHC, A05334 has pictured human, mouse, and rat samples and is polyclonal; M05334 is monoclonal, lists human and mouse reactivity, and pictures human kidney (catalog dilution notes, reactivity, and IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UI32 (GLSL_HUMAN, Glutaminase liver isoform, mitochondrial).
- Human Protein Atlas. GLS2 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. GLS2 subcellular location (ICC-IF): Highest expression in Hep-G2: 7.7 nTPM.
- Human Protein Atlas. GLS2 antibody validation summary (1 antibodies).
- Kidney-type glutaminase (GLS1) is a biomarker for pathologic diagnosis and prognosis of hepatocellular carcinoma. Oncotarget 2015 — PMC4480704.
- The metabolic shift of glutaminase 2 to glutaminase 1 promotes LGR5 + progenitor cell proliferation in liver cirrhosis. Cellular and molecular life sciences : CMLS 2025 — PMC12185844.
- Increased Glutaminolysis Marks Active Scarring in Nonalcoholic Steatohepatitis Progression. Cellular and molecular gastroenterology and hepatology 2020 — PMC7215180.
- Atractylenolide III Ameliorates Bile Duct Ligation-Induced Liver Fibrosis by Inhibiting the PI3K/AKT Pathway and Regulating Glutamine Metabolism. Molecules (Basel, Switzerland) 2023 — PMC10383814.
- PubMed PMID:10620514 — UniProt-cited evidence.
- PubMed PMID:12444921 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for GLS2 (Glutaminase liver isoform, mitochondrial). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); mitochondrial protein (UniProt). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A05334). Validated IHC antibody: A05334 at 0.5-2 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections. Protocol steps: 1. Sample: Formaldehyde-fixed, paraffin-embedded human liver tissue (datasheet A05334); 2. Fixation: Image formalin-fixed; duration unreported (datasheet A05334); 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: Citrate pH 6 (datasheet A05334); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% serum (datasheet A05334); 8. Primary antibody: Rabbit anti-GLS2, 0.5-2 μg/mL (datasheet A05334); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Use formaldehyde-fixed paraffin sections (selected-SKU IHC image A05334); keep fixation consistent (standard IHC practice; not target-specific); Caveat: Presumed off-target binding; verify specificity (HPA tissue IHC); Isoform / epitope: Two isoforms ; map epitopes to mature chain 15–602 (UniProt); Regulation: Reduced in hepatocellular carcinomas (UniProt). Evidence: UniProt Q9UI32, HPA tissue IHC · Uncertain, HPA cell-line RNA · Highest expression in Hep-G2: 7.7 nTPM, Datasheet A05334, PMC4480704, PMC7215180. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/GLS2/immunohistochemistry-ihc-guide.