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ATG4C / Cysteine protease ATG4C · IHC design guide
Design Immunohistochemistry for ATG4C
Plan ATG4C paraffin IHC around cytoplasmic staining (HPA tissue IHC), using catalog antibody A09728 at a starting concentration of 5 μg/mL (datasheet). Interpret staining intensity cautiously because antibody staining and RNA expression show low consistency (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ATG4C 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) | |
| Staining pattern | General cytoplasmic staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show low consistency (HPA tissue IHC) | |
| Regulation | No induction pattern reported (UniProt) | |
| Isoform / epitope | No isoforms annotated; one 1–458 chain (UniProt) |
Section 1
Recommended ATG4C IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet) is accompanied by 3 published ATG4C IHC protocols (PMC4369745; PMC6617611; PMC7219755).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A09728); 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-ATG4C, 5 μg/mL (datasheet A09728) |
| 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 | ATG4C-positive staining in adipocytes of adipose tissue (HPA tissue IHC: Medium). HPA tissue profile: General cytoplasmic expression. 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; UniProt Q96DT6: cytoplasm).
Section 2
What Is the Expected ATG4C Staining Pattern?
ATG4C is annotated as cytoplasmic and has no transmembrane segment (UniProt Q96DT6). In paraffin sections, expect mainly cytoplasmic staining in cells with reported medium signal, such as adipocytes, adrenal glandular cells and colon endothelial cells (HPA tissue IHC). HPA describes general cytoplasmic expression, but rates its IHC evidence Approved with low agreement between staining and RNA data, a transcript discrepancy, and pending external verification (HPA tissue IHC).
| Cytoplasmic staining in adipocytes, adrenal glandular cells or colon endothelial cells (HPA tissue IHC). | These cell types have reported medium staining, consistent with HPA's general cytoplasmic profile and UniProt's cytoplasmic annotation (HPA tissue IHC; UniProt Q96DT6). Compare cells within the section before assigning a score; medium describes the reported HPA result, not a guaranteed intensity in every specimen (HPA tissue IHC; general IHC practice). |
| Strong staining confined to the plasma membrane or extracellular space (UniProt Q96DT6 topology). | That distribution does not fit the annotated cytoplasmic location or absence of a transmembrane segment (UniProt Q96DT6). Treat an isolated membrane or extracellular pattern as a possible artefact; inspect morphology and the detection controls before calling it ATG4C (general IHC practice). |
| Strong staining in a cell type reported as low, such as lung alveolar cells (HPA tissue IHC). | A strong result conflicts with that reported low pattern but does not prove cross-reactivity: low is not absent, and HPA flags limited staining–RNA agreement (HPA tissue IHC). Check cell identity and compare a reagent control; cross-reactivity or endogenous detection activity can create misleading staining (general IHC practice). |
| A diffuse haze covers cells and surrounding tissue, obscuring cell boundaries (general IHC practice). | Uninterpretable background cannot establish cytoplasmic ATG4C, even in a tissue with reported medium staining (HPA tissue IHC; general IHC practice). Uneven reagent deposition, incomplete blocking or excessive detection signal are general possibilities to check with the same processing and detection controls (general IHC practice). |
| No discernible signal in adrenal glandular cells or adipocytes (HPA tissue IHC). | HPA reports medium staining in those cells, so an entirely blank result warrants a workflow check (HPA tissue IHC; general IHC practice). It does not by itself establish absent ATG4C: the reference is Approved with low staining–RNA agreement and pending external verification (HPA tissue IHC). |
💡Expected ATG4C appearanceCall a result supportive when predominantly cytoplasmic staining is discernible in a reported medium cell population, such as adipocytes, with interpretable cell boundaries (HPA tissue IHC; UniProt Q96DT6; general IHC practice); isolated strong membrane or extracellular staining is suspect (UniProt Q96DT6 topology; general IHC practice).
| Compartment and topology (UniProt Q96DT6) | UniProt places ATG4C in the cytoplasm and reports no transmembrane segment (UniProt Q96DT6). Use cytoplasmic distribution as the main IHC pattern check; topology alone cannot identify the stained protein or establish antibody specificity (UniProt Q96DT6; general IHC practice). |
| Tissue and cell context (HPA tissue IHC) | HPA reports medium staining across several cell types, including neuronal cells in cerebral cortex and hematopoietic cells in bone marrow, and low staining in lung alveolar cells (HPA tissue IHC). Score the identified cell population, because a whole-section average can hide that distinction (general IHC practice). |
| Strength of IHC evidence (HPA tissue IHC; HPA antibodies) | The tissue profile is Approved, yet HPA reports low staining–RNA consistency, a splice or transcript discrepancy, and pending external verification (HPA tissue IHC). HPA007049 is listed as IHC Approved; the supplied antibody record does not list an IHC Enhanced designation (HPA antibodies). |
| IF/ICC location (HPA subcellular ICC-IF) | Q: Can nuclear fluorescence be expected in IF/ICC? A: HPA approves both nucleoplasm and cytosol for its ICC-IF location summary (HPA subcellular ICC-IF). That finding is useful context for an IHC nuclear signal, but does not establish a nuclear IHC pattern in paraffin sections (HPA subcellular ICC-IF; HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| The section is blank, including a cell population reported as medium (HPA tissue IHC). | The run or detection may have failed; a medium HPA result is a reference pattern, not a promise for every specimen (HPA tissue IHC; general IHC practice). | Check section integrity, primary antibody application, detection reagents and a concurrent positive control; then reassess the named cell population (general IHC practice). |
| Only membrane or extracellular staining is conspicuous (UniProt Q96DT6 topology). | The pattern conflicts with cytoplasmic annotation and no transmembrane segment; artefactual deposition or nonspecific detection is possible (UniProt Q96DT6; general IHC practice). | Inspect morphology and compare a no-primary control processed with the same detection reagents; withhold a positive call if the discordant pattern persists (general IHC practice). |
| Cells reported as low stain much more strongly than reported medium cells (HPA tissue IHC). | Cell identification, cross-reactivity or endogenous detection activity may explain the mismatch; HPA's tissue profile also has low staining–RNA agreement (general IHC practice; HPA tissue IHC). | Recheck cell identity and compare primary-omission and detection controls before scoring the result as ATG4C (general IHC practice). |
| Diffuse color obscures cytoplasm and tissue structure (general IHC practice). | Excess background can prevent a compartment call; insufficient blocking or excessive detection signal are general IHC possibilities (general IHC practice). | Review blocking, wash and detection conditions with a no-primary control, then score only sections where cell boundaries are readable (general IHC practice). |
| Nuclear staining accompanies cytoplasmic staining in IHC (HPA tissue IHC; HPA subcellular ICC-IF). | HPA reports approved nucleoplasmic and cytosolic locations in ICC-IF, while its tissue IHC profile describes general cytoplasmic expression (HPA subcellular ICC-IF; HPA tissue IHC). | Record nuclear and cytoplasmic staining separately; check controls and avoid treating the ICC-IF observation as proof of nuclear IHC specificity (general IHC practice; HPA subcellular ICC-IF). |
| Staining varies between sections or tissue regions (general IHC practice). | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. | Compare equivalent cell populations and concurrent controls under the same processing conditions; report uninterpretable areas separately (general IHC practice). |
Experiment evidence
IHC & IF images showing ATG4C staining
A09728 · IHCImmunohistochemistry of ATG4C in human lung tissue with ATG4C antibody at 5 μg/ml.
A09728 · IFImmunofluorescence of ATG4C in human lung tissue with ATG4C antibody at 20 μg/ml.
A09728-2 · IFIF analysis of ATG4C using anti-ATG4C antibody (A09728-2) and anti-Tubulin Alpha antibody (M03989-3). ATG4C was detected in immunocytochemic
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000125703-ATG4C/tissue/cerebral+cortex#imid_2590208
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000125703-ATG4C/tissue/colon#imid_2590134
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000125703-ATG4C/tissue/liver#imid_2590127
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000125703-ATG4C/tissue/kidney#imid_2590136
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA007049; green = ATG4C, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA007049; green = ATG4C, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ATG4C IHC & IF
🧪Run bone marrow first and assess staining in hematopoietic cells (HPA: Medium in bone marrow hematopoietic cells). HPA detects ATG4C in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and treat any unstained cells on the positive slide as background comparators rather than validated internal negatives (HPA: no negative tissue rows; standard IHC practice).
Positive control tissue: Adipose tissue (Adipocytes, HPA Medium)
Negative control tissue: None in HPA: ATG4C is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ATG4C in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only section, a host-species- and clonality-matched isotype control, and ATG4C-knockout material as a biological negative where available (standard IHC practice). Block endogenous peroxidase and inspect the bone marrow control for residual peroxidase signal before interpreting chromogenic staining (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A09728 lung IHC caption reports 5 µg/ml but leaves the fixative unreported (selected tissue-IHC caption: A09728). Antigen retrieval dependence and any advantage of frozen sections are unreported in the supplied evidence (supplied target/application evidence). IF/ICC is feasible to assess separately because HPA provides images in A-431, U-251MG and U2OS cells; for the proposed bone marrow IHC control, endogenous peroxidase is a relevant artefact to check (HPA: subcellular ICC-IF images; standard IHC practice).
HPA tissue IHC evidence for ATG4C
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. Caution, Splice and/or transcript discrepancy exists. 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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: ATG4C is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced ATG4C IHC Tips
Use matched controls and compartment-aware scoring when troubleshooting ATG4C staining in paraffin-section IHC (UniProt Q96DT6; standard IHC practice).
How should I adjust retrieval when ATG4C staining is weak?
Begin with citrate buffer at pH 6.0, 95–98 °C for 20 min (page retrieval protocol). Let sections cool in buffer, then rinse and compare matched control sections processed in the same run (standard IHC practice). ATG4C is cytoplasmic and has no transmembrane segment (UniProt Q96DT6), so assess cellular staining without expecting a sharp membrane outline. If signal remains weak, check deparaffinization, antibody concentration and detection chemistry; the selected human lung image used 5 µg/ml of antibody (A09728 caption). Test any alternate retrieval condition on paired sections and record both morphology and background (standard IHC practice).
Could fixation explain inconsistent ATG4C staining between paraffin blocks?
Target-specific sensitivity of ATG4C to fixation is unknown from the supplied evidence; the selected human lung caption does not state its fixative (A09728 caption). Record fixative, fixation duration, processing schedule and block age for each specimen before comparing staining (standard IHC practice). Run sections from differently processed blocks together with identical pH 6.0, 95–98 °C, 20 min retrieval and detection settings (page retrieval protocol; standard IHC practice). Compare preserved cellular morphology and control staining alongside ATG4C intensity, because processing differences can affect IHC generally (standard IHC practice). Do not assign an ATG4C-specific fixation effect without a controlled comparison (standard IHC practice).
Should ATG4C staining be cytoplasmic, nuclear or membranous?
Expect a cytoplasmic component in tissue IHC: ATG4C is annotated in the cytoplasm, and tissue staining is described as generally cytoplasmic (UniProt Q96DT6; HPA tissue IHC). Nucleoplasmic and cytosolic localisation are both approved in the separate subcellular imaging record, so nuclear signal merits evaluation rather than automatic rejection (HPA subcellular). A crisp cell-surface rim is less consistent with a protein lacking a transmembrane segment (UniProt Q96DT6). Compare each compartment against no-primary and matched tissue controls, while checking whether pigment or counterstain accounts for apparent nuclear colour (standard IHC practice). Record cytoplasmic and nuclear scores separately instead of combining unlike patterns (standard IHC practice).
How can epitope choice complicate ATG4C IHC interpretation?
The supplied UniProt record lists a single 1–458 chain and no annotated isoforms, propeptide or transmembrane segment (UniProt Q96DT6). It also annotates N-terminal acetylation and phosphorylation at residues 451 and 452; the antibody epitope is not supplied (UniProt Q96DT6; supplied antibody evidence). Check the antibody's documented immunogen or epitope before attributing variable staining to a particular terminal modification (standard IHC practice). If that information is unavailable, compare an independently characterised antibody directed to another region on adjacent sections, using matched retrieval and scoring (standard IHC practice). Concordance supports an ATG4C-associated pattern but does not establish modification-specific detection (standard IHC practice).
How should I assess an ATG4C pattern by multiplex IF?
Treat IF as a separate assay requiring its own antibody and sample validation; the selected ATG4C caption documents tissue IHC only (A09728 caption). Multiplex with an independently validated marker identifying the cell type being scored, such as adipocytes in adipose tissue, where ATG4C staining was rated medium (HPA tissue IHC; standard IF practice). Choose fluorophores after examining unstained tissue autofluorescence, favouring channels with a clear signal over the local background (standard IF practice). For an accessible cytosolic epitope, use a controlled post-fixation permeabilisation step; ATG4C has no transmembrane segment, but this antibody's epitope is unspecified (UniProt Q96DT6; standard IF practice). Include single-stain and no-primary controls when assessing overlap (standard IF practice).
What should I check when ATG4C DAB staining looks diffuse?
Compare the stained slide with no-primary and detection-only controls to separate antibody signal from nonspecific reagent deposition (standard IHC practice). Confirm adequate deparaffinization, washing, protein blocking and endogenous peroxidase blocking before interpreting diffuse DAB colour (standard IHC practice). ATG4C has a general cytoplasmic tissue staining profile, so diffuse cellular staining alone is insufficient proof of specificity (HPA tissue IHC; standard IHC practice). Titrate antibody and detection exposure on adjacent sections while preserving the same retrieval setting of citrate pH 6.0 for 20 min (page retrieval protocol; standard IHC practice). Record whether background concentrates at tissue edges, damaged areas or extracellular spaces (standard IHC practice).
How should I score ATG4C across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and compartment before reading slides, then apply the same thresholds across the staining batch (standard IHC practice). For cellular expression, report an H-score using intensity categories and the percentage of cells in each category, or report percentage positive cells with a fixed threshold (standard IHC practice). Normalise counts to eligible intact cells; if measuring positive-cell density, report cells per mm² of evaluable tissue and exclude empty or necrotic areas (standard IHC practice). Score cytoplasmic and nuclear signal separately because tissue IHC describes general cytoplasmic expression while subcellular imaging also supports nucleoplasmic localisation (HPA tissue IHC; HPA subcellular). Include control-slide performance with each batch (standard IHC practice).
How can I distinguish genuine ATG4C staining from artefact?
A plausible result includes cellular cytoplasmic staining; nucleoplasmic signal may also be plausible, whereas a dominant sharp membrane pattern needs additional validation (UniProt Q96DT6; HPA tissue IHC; HPA subcellular). Check the identity of stained cells against morphology and an appropriate marker rather than assuming every positive cell belongs to the expected population (standard IHC practice). Discount edge-concentrated colour, necrotic regions and signal reproduced in no-primary or peroxidase-control sections (standard IHC practice). The human lung caption shows staining at 5 µg/ml, but HPA rates alveolar-cell staining low, so that image alone cannot establish broad lung positivity (A09728 caption; HPA tissue IHC). Interpret discordant cases cautiously because HPA reports low agreement between staining and RNA data (HPA tissue IHC).
Boster reagents
Best ATG4C / Cysteine protease ATG4C IHC Antibodies
A09728 has human lung tissue IHC and IF images (catalog image captions) and lists Human, Mouse and Rat reactivity (catalog reactivity); A09728-2 shows IF in U20S cells (catalog image caption).
Real IHC data
Anti-Cysteine protease ATG4C ATG4C Antibody
Cat # A09728
Real IF data
Anti-ATG4C Antibody ®
Cat # A09728-2
A09728 lists IHC-P and IF, with images of ATG4C staining in human lung tissue for both applications (catalog applications; image captions). A09728-2 lists IF and ICC, with an IF image from U20S cells (catalog applications; image caption).
Which to pick: For tissue IHC, choose A09728: it lists IHC-P and shows human lung tissue stained at 5 μg/mL; the image caption does not report the fixative (catalog applications; A09728 IHC image caption). For cell IF/ICC, choose A09728-2, which lists both applications and shows IF in U20S cells (catalog applications; A09728-2 IF image caption). For work across species, A09728 lists Human, Mouse and Rat reactivity, although its IHC image shows human tissue only; clonality is unreported for both antibodies (catalog reactivity; A09728 IHC image caption; catalog clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q96DT6 (ATG4C_HUMAN, Cysteine protease ATG4C).
- Human Protein Atlas. ATG4C tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ATG4C subcellular location (ICC-IF): Localized to the nucleoplasm and cytosol..
- Human Protein Atlas. ATG4C antibody validation summary (1 antibodies).
- Altered autophagic flux enhances inflammatory responses during inflammation-induced preterm labor. Scientific reports 2015 — PMC4369745.
- Exploring the interplay between EBV and autophagy-related gene expression patterns in nasopharyngeal carcinoma. Frontiers in oncology 2025 — PMC12234470.
- Knockdown ATG4C inhibits gliomas progression and promotes temozolomide chemosensitivity by suppressing autophagic flux. Journal of experimental & clinical cancer research : CR 2019 — PMC6617611.
- Clinicopathological and prognostic significance of caveolin-1 and ATG4C expression in the epithelial ovarian cancer. PloS one 2020 — PMC7219755.
- PubMed PMID:12446702 — UniProt-cited evidence.
- PubMed PMID:30808002 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ATG4C (Cysteine protease ATG4C). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A09728 at 5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. 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 A09728); 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-ATG4C, 5 μg/mL (datasheet A09728); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA show low consistency (HPA tissue IHC); Isoform / epitope: No isoforms annotated; one 1–458 chain (UniProt); Regulation: No induction pattern reported (UniProt). Evidence: UniProt Q96DT6, HPA tissue IHC · Approved, HPA ICC-IF · nucleoplasm and cytosol., Datasheet A09728, PMC4369745, PMC6617611. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATG4C/immunohistochemistry-ihc-guide.