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ATG4C / Cysteine protease ATG4C · IHC design guide
Design Immunohistochemistry for ATG4C
Plan ATG4C paraffin IHC around general cytoplasmic staining (HPA tissue IHC). Start catalog antibody A09728 at 5 μg/mL (datasheet), and interpret intensity cautiously because tissue staining and RNA 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 staining in tissue (HPA tissue IHC) | |
| Staining pattern | General cytoplasmic staining (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). Selected-image fixative and duration unreported (datasheet A09728); verify before use. | |
| Caveat | Antibody staining and RNA show low consistency (HPA tissue IHC) | |
| Regulation | No tissue regulation annotated (UniProt) | |
| Isoform / epitope | No isoforms or processing variants annotated (UniProt) |
Section 1
Recommended ATG4C IHC & IF Protocols
The catalog antibody IHC-P protocol is followed by four published ATG4C IHC workflows (PMC4369745; PMC12234470; 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 noteFor cytoplasmic ATG4C (UniProt Q96DT6), start with citrate HIER at pH 6.0, 95–98 °C for 20 min (page antigen retrieval).
Section 2
What Is the Expected ATG4C Staining Pattern?
Expect ATG4C staining chiefly in the cytoplasm of cells with an HPA IHC signal, including adipocytes, glandular cells, hematopoietic cells, neurons and endothelial cells (HPA tissue IHC: general cytoplasmic expression; listed cell types). ATG4C is cytoplasmic and has no annotated transmembrane segment (UniProt Q96DT6: location and topology). Treat the pattern as provisional: HPA rates its IHC evidence Approved but reports low agreement with RNA data and pending external verification (HPA tissue IHC: reliability).
| Cytoplasmic chromogen signal in adipocytes or listed glandular, hematopoietic, neuronal or endothelial cells. | This fits the reported compartment and cell distribution. HPA records Medium staining in these listed examples, so score the named cells rather than calling every cell in the section positive (HPA tissue IHC: profile and positive entries; UniProt Q96DT6: cytoplasm). |
| Predominantly nuclear-only, surface-rim or extracellular signal in an IHC section. | This is discordant with the reported cytoplasmic IHC pattern and warrants an artefact check (HPA tissue IHC: profile; UniProt Q96DT6: topology). Nuclear signal needs assay context: HPA separately approves nucleoplasmic localisation by ICC-IF (HPA subcellular: approved locations). |
| Strong colour in cells outside the reported stained cell populations, especially where expected cells remain clear. | Consider cross-reactivity or endogenous detection activity before assigning ATG4C to those cells (general IHC practice). HPA lists specific stained cell types and cautions that its antibody staining has low agreement with RNA data (HPA tissue IHC: positive entries and reliability). |
| Diffuse colour across stroma, empty spaces and many unrelated cell types. | A nonselective background pattern does not establish ATG4C localisation. Check the detection controls and compare cellular signal with background using the same scoring threshold (general IHC practice); the reported IHC profile is cytoplasmic (HPA tissue IHC: profile). |
| No cellular signal in a section chosen from an HPA Medium-staining tissue. | First assess assay performance with the reported positive cell population and controls (general IHC practice). Absence alone is inconclusive because HPA's IHC pattern has low agreement with RNA data and awaits external verification (HPA tissue IHC: reliability). |
💡Expected ATG4C appearanceCall a section consistent with ATG4C when its listed cell population shows discernible, mainly cytoplasmic staining around the reported Medium level; dominant nuclear-only, surface-rim or diffuse extracellular colour is discordant with the IHC profile (HPA tissue IHC: profile and positive entries; UniProt Q96DT6: topology).
| Compartment and topology | UniProt places ATG4C in the cytoplasm and annotates no transmembrane segment; HPA describes general cytoplasmic IHC expression (UniProt Q96DT6: location and topology; HPA tissue IHC: profile). Evaluate signal within cells rather than expecting a cell-surface outline. |
| Choice of reference tissue and cells | HPA reports Medium staining in, among others, adipocytes, adrenal glandular cells, bone marrow hematopoietic cells, cortical neurons and colon endothelial cells (HPA tissue IHC: positive entries). Its Low entries include lung alveolar cells and bronchial respiratory epithelium (HPA tissue IHC: low entries). |
| Strength of the tissue evidence | HPA calls the IHC result Approved, while flagging low agreement between antibody staining and RNA expression, a splice or transcript discrepancy, and pending external verification (HPA tissue IHC: reliability). Treat its intensity map as a comparison point, not a definitive expression boundary. |
| Antibody validation scope | The supplied HPA record lists one rabbit polyclonal antibody, HPA007049, as Approved for IHC and ICC; it does not list an Enhanced IHC result (HPA antibodies: HPA007049). A matching image alone therefore cannot establish that every stained cell contains ATG4C. |
| IF/ICC Q&A: is nuclear staining expected? | For ICC-IF, HPA approves both nucleoplasm and cytosol and lists images from A-431, U-251MG and U2OS (HPA subcellular: approved locations and image lines). For paraffin IHC, use the separately reported general cytoplasmic profile when interpreting a section (HPA tissue IHC: profile). |
| Situation | Likely cause | Next action |
|---|---|---|
| A reference section is blank. | The assay may have failed, or the sampled area may lack the HPA-listed positive cells; a blank section alone cannot distinguish these possibilities (general IHC practice; HPA tissue IHC: positive entries). | Locate the reported cell population, review counterstain and detection controls, then repeat with an HPA Medium-staining tissue if needed (general IHC practice; HPA tissue IHC: positive entries). |
| Only nuclei stain in paraffin IHC. | This conflicts with HPA's general cytoplasmic IHC profile, although HPA separately approves nucleoplasmic ICC-IF localisation (HPA tissue IHC: profile; HPA subcellular: approved locations). | Check control sections and whether colour is genuinely intranuclear before assigning an IHC result; record the assay-specific difference (general IHC practice; HPA subcellular: ICC-IF locations). |
| Unexpected cells stain more strongly than the reported population. | Cross-reactivity or endogenous detection activity is possible (general IHC practice); HPA also warns of low agreement between staining and RNA data (HPA tissue IHC: reliability). | Compare the same cell types across appropriate reagent controls and an HPA-listed tissue, then report any discordance rather than extending the cell-type claim (general IHC practice; HPA tissue IHC: positive entries). |
| The whole section has diffuse chromogen. | Nonselective reagent binding or endogenous detection activity can obscure cellular localisation (general IHC practice). This appearance differs from the reported general cytoplasmic profile (HPA tissue IHC: profile). | Review blocking, washes and detection controls, and assess whether cell-associated signal remains distinguishable from background (general IHC practice). |
| Dark staining obscures cell boundaries. | Excess primary antibody or chromogen development can reduce readable contrast (general IHC practice); the reported HPA positives are described as Medium, not as a required saturation level (HPA tissue IHC: positive entries). | Optimize the catalog antibody's IHC-P dilution and development time using its IHC instructions, then score discernible cytoplasmic signal in identified cells (general IHC practice; HPA tissue IHC: profile). |
| A Low-staining cell population appears negative. | A weak or absent visible signal is compatible with a Low HPA entry; examples include lung alveolar cells and parathyroid glandular cells (HPA tissue IHC: low entries). | Check an HPA Medium-staining cell population in parallel before judging assay failure, and report the Low population separately (general IHC practice; HPA tissue IHC: positive and low entries). |
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 adipose tissue first and look for staining in adipocytes (HPA: Medium in adipocytes). HPA lists no negative tissue because ATG4C is detected in all 45 scored tissues, so use no-primary and isotype controls for background; cells without visible staining on the positive slide should show only counterstain or background and are not validated biological negatives (HPA: detected in all 45 scored tissues; standard IHC interpretation).
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 control, a control antibody matched to the primary antibody’s host species and clonality, and ATG4C knockout material if available; peptide blocking can provide an additional specificity check (standard IHC control practice). For chromogenic detection in adipose tissue, quench endogenous peroxidase and distinguish staining in the thin adipocyte cytoplasm from empty lipid spaces (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the selected A09728 lung IHC caption does not state a fixative (A09728 tissue-IHC caption). Antigen retrieval dependence is unreported, so optimize retrieval on the positive tissue (standard IHC practice). HPA has ICC-IF images and reports nucleoplasm and cytosol localization, but the supplied evidence does not establish whether IF or frozen sections are easier than paraffin IHC; adipocyte lipid spaces can complicate scoring (HPA: approved nucleoplasm and cytosol; 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 compartment, cell type and control stains to evaluate ATG4C signal; the supplied tissue and subcellular references warrant careful interpretation (UniProt Q96DT6; HPA tissue IHC; HPA subcellular).
How should I retrieve ATG4C in paraffin sections when staining is weak?
Start with citrate pH 6.0 heat-induced retrieval at 95–98 °C for 20 min (page retrieval rule: cytoplasmic / membrane antigen). Let sections cool in buffer before washing, then compare a short antibody dilution series on adjacent sections while keeping detection conditions constant (standard IHC practice). The selected A09728 lung caption reports 5 µg/ml antibody but gives no retrieval method, so that image cannot establish an optimal retrieval condition (A09728 tissue-IHC caption). If cytoplasmic staining remains weak, compare a higher-pH retrieval buffer on matched sections and retain the condition that improves cellular signal without increasing tissue-edge staining (standard IHC practice; UniProt Q96DT6: cytoplasm).
How can I troubleshoot weak ATG4C staining after fixation?
ATG4C-specific fixation sensitivity is unknown because the supplied A09728 tissue-IHC caption does not state a fixative (A09728 tissue-IHC caption). Record the actual fixative, fixation interval and processing history, then compare adjacent sections processed alike before changing antibody concentration or retrieval (standard IHC practice). For newly prepared material, a documented 10% neutral-buffered formalin workflow provides a reproducible starting point, but it is a general tissue-processing choice rather than an ATG4C-validated condition (standard IHC practice). Include a matched reference section in each run, and interpret signal loss only after checking morphology, counterstain and a tissue control processed with the same fixation history (standard IHC practice).
Should nuclear or membrane staining count as ATG4C signal?
Score the cytoplasmic pattern first because UniProt lists ATG4C in the cytoplasm, while HPA reports approved cytosol and nucleoplasm locations in its subcellular assay (UniProt Q96DT6: cytoplasm; HPA subcellular: nucleoplasm and cytosol). ATG4C has no annotated transmembrane segment, so crisp cell-border staining alone needs independent support before being called target signal (UniProt Q96DT6: topology). Compare nuclear and cytoplasmic staining separately on serial sections, using the same counterstain and exposure to the chromogen across conditions (standard IHC practice). If only nuclei stain in paraffin sections, check a second antibody or orthogonal target measurement before equating that pattern with the HPA cell-image result (HPA subcellular: nucleoplasm; standard IHC practice).
Could epitope location explain discordant ATG4C stains?
Ask for the immunogen or epitope coordinates before interpreting discordant stains; none are provided for the selected antibody in this payload (A09728 tissue-IHC caption). The supplied UniProt record lists a single 1–458 chain and 0 annotated isoforms, which limits what can be inferred about isoform-selective staining from this record (UniProt Q96DT6: processing and isoforms). It also lists N-terminal acetylation and phosphorylation at residues 451 and 452, but their effect on this antibody's binding is unknown without epitope mapping (UniProt Q96DT6: modified residues; A09728 tissue-IHC caption). Compare independently mapped antibodies on serial sections and avoid assigning an epitope or splice form from chromogenic pattern alone (standard IHC practice).
How would I check an ATG4C IHC pattern by multiplex IF?
On a separately validated IF assay, pair ATG4C with an endothelial marker when examining colon, where HPA reports medium staining in endothelial cells (HPA tissue IHC: colon endothelial cells; standard IF practice). Choose a far-red ATG4C channel when the tissue has strong green autofluorescence, and include single-stain controls to assess spectral bleed-through (standard IF practice). Because ATG4C has no transmembrane segment and is reported in the cytosol, use a mild permeabilisation step to admit antibody to intracellular epitopes; optimise it against morphology and marker retention (UniProt Q96DT6: topology; HPA subcellular: cytosol; standard IF practice). Compare cytosolic and nuclear signal separately, since HPA also reports nucleoplasm staining, and do not transfer the A09728 tissue-IHC caption into an IF validation claim (HPA subcellular: nucleoplasm; A09728 tissue-IHC caption).
What causes diffuse brown background in ATG4C IHC?
Run a no-primary control alongside the catalog antibody and inspect whether brown signal follows cells, tissue folds or section edges (standard chromogenic IHC practice). Quench endogenous peroxidase before an HRP–DAB detection step, and use an appropriate protein block plus thorough washes to reduce nonspecific deposition (standard chromogenic IHC practice). If background rises with the antibody concentration, titrate below the 5 µg/ml used in the selected lung image while holding retrieval and chromogen development constant (A09728 tissue-IHC caption; standard IHC practice). Judge any remaining diffuse signal against the expected cellular distribution rather than treating every brown region as ATG4C, which UniProt places in cytoplasm (UniProt Q96DT6: cytoplasm; standard IHC practice).
How should I quantify ATG4C across heterogeneous paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring: HPA describes general cytoplasmic tissue expression, while its subcellular assay also reports nucleoplasm (HPA tissue IHC: profile; HPA subcellular: nucleoplasm and cytosol). For chromogenic sections, report the percentage of positive cells and an H-score from intensity categories 0–3, with each percentage calculated within the prespecified cell type (standard IHC scoring practice). If cell counts vary substantially, report positive-cell density per mm² of viable tissue as a separate measure and record the area excluded for folds or necrosis (standard digital pathology practice). Keep retrieval, DAB development and image-analysis thresholds fixed, and normalise comparisons to viable area or the number of eligible cells rather than total slide area (standard IHC practice).
How can I distinguish credible ATG4C staining from artefact?
A credible result should show a reproducible cellular pattern with a cytoplasmic component, consistent with UniProt's cytoplasm annotation and HPA's general cytoplasmic tissue profile (UniProt Q96DT6: cytoplasm; HPA tissue IHC: profile). Check cell identity: HPA reports medium endothelial-cell staining in colon but low alveolar-cell staining in lung, so an A09728 lung image alone cannot establish strong alveolar expression (HPA tissue IHC: colon and lung; A09728 tissue-IHC caption). Exclude staining confined to section edges, folds or necrotic regions, and check a no-primary control for endogenous enzyme or detection-system signal (standard chromogenic IHC practice). Treat discrepancies cautiously because HPA labels tissue reliability Approved while noting low agreement with RNA and a possible splice or transcript discrepancy (HPA tissue IHC: reliability description).
Boster reagents
Best ATG4C / Cysteine protease ATG4C IHC Antibodies
A09728 has human lung IHC and IF images (catalog image captions); A09728-2 has a human U20S IF/ICC image (catalog IF image caption; catalog reactivity).
Real IHC data
Anti-Cysteine protease ATG4C ATG4C Antibody
Cat # A09728
Real IF data
Anti-ATG4C Antibody ®
Cat # A09728-2
A09728 will render with human lung IHC data at 5 μg/mL and has a human lung IF image (A09728 image captions). A09728-2 will render with IF/ICC data from a U20S cell at 5 μg/mL (A09728-2 IF image caption).
Which to pick: Choose A09728 for paraffin-section IHC: IHC-P is listed, and its own IHC caption shows human lung staining at 5 μg/mL; the fixative is unreported (A09728 catalog applications; A09728 IHC image caption). Choose A09728-2 for human IF/ICC because both applications are listed and its own image shows U20S cells (A09728-2 catalog applications; A09728-2 IF image caption; catalog reactivity). For mouse or rat work, A09728 lists reactivity with both species, while A09728-2 lists human only; both have a rabbit host and no clone stated (catalog reactivity; catalog host; catalog clone).
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 staining in tissue (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: 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). Selected-image fixative and duration unreported (datasheet A09728); verify before use.; Caveat: Antibody staining and RNA show low consistency (HPA tissue IHC); Isoform / epitope: No isoforms or processing variants annotated (UniProt); Regulation: No tissue regulation annotated (UniProt). Evidence: UniProt Q96DT6, HPA tissue IHC · Approved, HPA ICC-IF · nucleoplasm and cytosol., Datasheet A09728, PMC4369745, PMC12234470. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATG4C/immunohistochemistry-ihc-guide.