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ATG4D / Cysteine protease ATG4D · IHC design guide
Design Immunohistochemistry for ATG4D
Plan ATG4D chromogenic IHC in paraffin sections using its mainly cytoplasmic tissue pattern and strongly stained glandular and Leydig cells as reference points (HPA tissue IHC). The guide covers fixation, staining controls and interpretation of CASP3-dependent mitochondrial import (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ATG4D 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 | Mainly cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Glandular and Leydig cells: mainly cytoplasmic (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Duodenum+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep tissue fixation consistent. (standard IHC practice; not target-specific) | |
| Caveat | CASP3 cleavage can shift ATG4D to mitochondria (UniProt) | |
| Regulation | Expression regulation is unreported (UniProt) | |
| Isoform / epitope | 2 isoforms; cleavage removes residues 1–63, so check epitope (UniProt) |
Section 1
Recommended ATG4D IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet) is accompanied by three published ATG4D IHC protocols (PMC12397559; PMC5554887; PMC4398399).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A09793); 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-ATG4D, 5 μg/mL (datasheet A09793) |
| 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 | ATG4D-positive staining in glandular cells of duodenum (HPA tissue IHC: High). HPA tissue profile: Mainly cytoplasmic expression in several different tissue types. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen retrieval); use the cited conditions when reproducing a published study.
Section 2
What Is the Expected ATG4D Staining Pattern?
In paraffin sections, ATG4D staining is mainly cytoplasmic, with High staining in duodenal and small-intestinal glandular cells and testicular Leydig cells (HPA: tissue IHC). ATG4D has no transmembrane segment and can enter the mitochondrial matrix after CASP3 cleavage during oxidative stress or cell death (UniProt Q86TL0). HPA rates its tissue IHC profile Approved, with medium consistency between staining and RNA expression (HPA: tissue IHC).
| Cytoplasmic staining in intestinal glandular cells or testicular Leydig cells (HPA: High in these cells). | This matches the reported tissue pattern (HPA: mainly cytoplasmic; High in these cells). Compare intensity within the same section and tissue compartment; a dark chromogenic deposit alone does not establish mitochondrial localisation (general IHC practice). |
| Predominantly nuclear staining in a paraffin section (HPA: tissue IHC is mainly cytoplasmic). | Treat this as a mismatch requiring review, rather than proof of artefact: ICC-IF also reports nucleoplasm (HPA: nucleoplasm approved). Check whether cytoplasmic staining is present in an expected cell population and whether controls show nuclear background (general IHC practice). |
| Strong staining in adipocytes or muscle cells (HPA: Not detected in adipocytes, cardiomyocytes and myocytes). | This conflicts with those HPA tissue observations; possible explanations include cross-reactivity or endogenous detection activity (general IHC practice). Compare a known-positive cell population and a no-primary control before assigning the signal to ATG4D. |
| Uniform deposit across cells and extracellular spaces, with little cellular definition. | That distribution is difficult to reconcile with mainly cytoplasmic tissue staining (HPA: tissue IHC profile). Review blocking, washes and chromogen development against a no-primary control (general IHC practice); do not score diffuse deposit as positive cells. |
| No discernible staining in duodenal glandular cells or testicular Leydig cells (HPA: High in both). | A negative result in these expected-positive populations leaves assay performance unresolved; it does not establish biological absence (HPA: High in these cells). Check section quality, retrieval, primary-antibody application and detection controls (general IHC practice). |
💡Expected ATG4D appearanceCall a section positive when identifiable glandular cells or Leydig cells show mainly cytoplasmic staining, potentially High in the cited tissues (HPA: tissue IHC); widespread deposit without cell boundaries or strong signal in HPA-undetected cell populations warrants investigation (HPA: negative tissue observations; general IHC practice).
| Tissue and cell selection | Duodenum and small intestine glandular cells and testis Leydig cells are High, while several other reported populations are Medium, Low or Not detected (HPA: tissue IHC). Score the specified cell type, not the whole section. |
| IF/ICC Q&A: should nuclear or mitochondrial staining be expected? | ICC-IF reports nucleoplasm (approved) and mitochondria (supported) in U2OS images (HPA: subcellular). Paraffin tissue IHC is mainly cytoplasmic (HPA: tissue IHC); those assay observations should be interpreted in their own contexts. |
| Stress-associated processing | CASP3 cleavage can precede mitochondrial matrix import during oxidative stress and cell death (UniProt Q86TL0). Processing may alter localisation, but the supplied evidence gives no expected frequency or chromogenic appearance for that state in routine sections. |
| Isoforms and epitope coverage | UniProt lists 2 isoforms and full-length and 64–474 chains (UniProt Q86TL0). Without an antibody epitope or isoform-reactivity map in these payloads, neither relative staining nor recognition of the processed chain can be predicted. |
| Confidence in the tissue pattern | The listed antibody HPA067683 is Approved for IHC, while agreement between tissue staining and RNA expression is medium (HPA: antibody validation; tissue IHC). Use the reported pattern as a reference, and investigate discrepancies with controls. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected-positive glandular cells remain blank (HPA: High in duodenum and small intestine). | The result may reflect inadequate assay signal or a section problem; absence of staining alone cannot distinguish these causes (general IHC practice). | Check tissue preservation, retrieval execution, primary-antibody application and the detection system using an appropriate positive control (general IHC practice). |
| The entire section has a faint, even chromogenic haze. | Non-specific background or endogenous detection activity can obscure cell boundaries (general IHC practice). | Review the no-primary control, blocking, washes and chromogen development; score only identifiable cells with resolved localisation (general IHC practice). |
| Strong signal appears in adipocytes or skeletal myocytes (HPA: Not detected in these populations). | Cross-reactivity or endogenous activity is possible; the HPA observations alone do not identify which (HPA: tissue IHC; general IHC practice). | Compare a no-primary control and an expected-positive cell population on matched runs before calling the unexpected staining ATG4D (general IHC practice). |
| Staining is largely nuclear in tissue sections (HPA: tissue IHC mainly cytoplasmic). | The result differs from the tissue profile, although nucleoplasmic ICC-IF localisation is approved (HPA: subcellular). | Check cell boundaries and control staining, then report the tissue and assay context; do not infer that nuclear signal is artefactual solely from the tissue profile (general IHC practice; HPA: subcellular). |
| Punctate cytoplasmic staining is interpreted as mitochondrial ATG4D. | Chromogenic puncta alone cannot establish organelle identity (general IHC practice), even though mitochondrial localisation is reported (HPA: ICC-IF supported; UniProt Q86TL0). | Describe the visible punctate pattern without assigning an organelle; use the separate IF/ICC localisation evidence when discussing that possibility (HPA: subcellular; general IHC practice). |
| Replicate sections show inconsistent intensity in the same expected-positive cells (HPA: High in intestinal glandular cells and Leydig cells). | Differences in section handling, retrieval or detection may contribute (general IHC practice); the supplied sources do not establish ATG4D-specific fixation sensitivity. | Compare matched controls and processing records, then repeat under consistent IHC conditions before attributing the difference to ATG4D expression (general IHC practice). |
Experiment evidence
IHC & IF images showing ATG4D staining
A09793 · IHCImmunohistochemistry of ATG4D in human testis tissue with ATG4D antibody at 5 μg/ml.
A09793 · IFImmunofluorescence of ATG4D in human testis tissue with ATG4D antibody at 20 μg/ml.
M09793 · IFImmunofluorescent analysis of U251 cells, using ATG4D Antibody. U251 cells (right) were treated with Chloroquine (50 μM,16h). M09793 was dil
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000130734-ATG4D/tissue/cerebral+cortex#imid_19069087
HPA · DuodenumHuman Protein Atlas IHC, Duodenum. Source: https://www.proteinatlas.org/ENSG00000130734-ATG4D/tissue/duodenum#imid_19069012
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000130734-ATG4D/tissue/colon#imid_19069005
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000130734-ATG4D/tissue/liver#imid_19069022
HPA · ICC-IF U2OSHuman Protein Atlas immunofluorescence, U2OS, antibody HPA067683; green = ATG4D, blue = nucleus, red = microtubules.
HPA · ICC-IF U2OSHuman Protein Atlas immunofluorescence, U2OS, antibody HPA067683; green = ATG4D, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ATG4D IHC & IF
🧪Run testis first and expect staining in Leydig cells (HPA: High in testis Leydig cells; catalog antibody caption: testis IHC). Use heart muscle cardiomyocytes as a negative tissue (HPA: Not detected in heart muscle cardiomyocytes); on the testis slide, compare Leydig cell staining with morphologically distinct cells that remain at background, without assuming every other cell type is ATG4D-negative (HPA: High in Leydig cells only).
Positive control tissue: Duodenum (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ATG4D in U2OS, with annotated localisation: Nucleoplasm (approved), Mitochondria (supported) (HPA subcellular).
Technical controls: Include no-primary or secondary-only and host-species-matched isotype controls, using nonimmune IgG for a polyclonal primary, and validate specificity with ATG4D knockout material where available or peptide competition when the immunizing peptide is known (standard IHC practice). Block endogenous peroxidase for chromogenic IHC and inspect a no-primary testis section for pigment that could resemble chromogen; for IF, assess autofluorescence with an unstained section (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A09793 testis IHC caption does not state the fixative (catalog antibody caption: fixative unreported). Retrieval dependency is also unreported; compare retrieved and unretrieved paraffin sections during optimization (standard IHC practice). The supplied evidence does not establish whether frozen sections or IF are easier; Leydig cell pigment can complicate interpretation of brown chromogen in testis (standard histology/IHC practice).
HPA tissue IHC evidence for ATG4D
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data.). 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 |
|---|---|---|---|---|
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Small intestine | Glandular cells | High | Protein (IHC) | HPA → |
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ATG4D IHC Tips
Use compartment and cell-type controls when optimizing chromogenic ATG4D staining in paraffin sections; the selected antibody’s tissue caption does not report fixation.
What retrieval should I try first when ATG4D staining is weak in paraffin sections?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 minutes (page retrieval rule). Compare adjacent sections using identical antibody concentration, detection chemistry, and counterstain so retrieval is the main variable. Assess cytoplasmic staining in testicular Leydig cells or intestinal glandular cells as reference patterns (HPA: High in these cells). If staining remains weak, test an alternative retrieval buffer on adjacent sections as a fallback, checking for tissue damage and increased background. The selected testis caption reports 5 µg/ml antibody but gives no retrieval conditions (A09793 tissue-IHC caption).
How should I handle fixation when ATG4D signal varies between tissue blocks?
ATG4D-specific fixation sensitivity is unknown because the selected tissue-IHC caption does not state a fixative (A09793 tissue-IHC caption). Record each block’s fixative, fixation interval, processing history, and section age before attributing a signal difference to biology. Compare sections from similarly processed blocks with the same citrate pH 6.0 retrieval at 95–98 °C for 20 minutes (page retrieval rule). Inspect morphology and staining together; poor preservation or damaged section edges can make intensity comparisons unreliable. The published testis image supports staining at 5 µg/ml, but cannot establish a preferred fixation condition (A09793 tissue-IHC caption).
Should I accept nuclear or mitochondrial ATG4D staining in chromogenic IHC?
First score the predominant cytoplasmic pattern expected across tissues (HPA tissue IHC: mainly cytoplasmic expression). Mitochondrial matrix localisation is also annotated after CASP3 cleavage during oxidative stress and cell death (UniProt Q86TL0: subcellular location and processing). Nucleoplasmic and mitochondrial signals have been reported by cell imaging, but their appearance in chromogenic tissue sections needs independent validation (HPA subcellular: nucleoplasm approved, mitochondria supported). Record nuclear, diffuse cytoplasmic, and granular cytoplasmic staining separately, alongside cell identity and tissue condition. If a compartment dominates unexpectedly, compare matched controls and exclude edge staining before assigning it to ATG4D.
Could isoforms or proteolytic processing change what the ATG4D antibody detects?
ATG4D has 2 annotated isoforms, but the supplied antibody evidence does not map its epitope to either isoform (UniProt Q86TL0: isoforms; A09793 tissue-IHC caption). Its annotated chains span residues 1–474 and 64–474, with the shorter mitochondrial form arising after CASP3 cleavage (UniProt Q86TL0: processing). Therefore, do not infer which form produces a chromogenic signal without an antibody epitope map or orthogonal validation. A phosphoserine is annotated at residue 467, but no supplied evidence shows that it changes IHC recognition (UniProt Q86TL0: modified residues). Document staining by compartment and avoid interpreting intensity as an isoform ratio.
How can IF help resolve an ambiguous ATG4D IHC localisation pattern?
Use IF on a matched specimen to examine whether the chromogenic pattern corresponds to intracellular ATG4D, while treating the assays as separate validations. Multiplex ATG4D with a marker identifying the expected cell population, such as Leydig cells in testis or glandular cells in intestine (HPA tissue IHC: High in these cells). Choose a fluorophore channel after checking tissue autofluorescence and include single-stain controls for bleed-through. Apply permeabilisation sufficient to expose intracellular epitopes; ATG4D has no annotated transmembrane segment, and its exact antibody epitope is unspecified (UniProt Q86TL0: topology; A09793 tissue-IHC caption). Compare nuclear and mitochondrial patterns cautiously with the cell-imaging report (HPA subcellular: nucleoplasm approved, mitochondria supported).
How do I troubleshoot diffuse brown staining in an ATG4D IHC section?
Run a no-primary control through the same chromogenic detection steps to identify background from secondary reagents or endogenous enzyme activity. Include a peroxidase block before a peroxidase-based DAB reaction, and check whether residual brown signal persists in the control. Titrate the primary antibody around the reported 5 µg/ml testis-image concentration without treating that image as an optimization series (A09793 tissue-IHC caption). Compare background in cells reported as not detected, such as adipocytes, with positive-reference cells such as Leydig cells (HPA tissue IHC: adipocytes Not detected; Leydig cells High). Review section edges and damaged areas separately from intact tissue.
What should I score when comparing ATG4D IHC across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring; ATG4D tissue staining is mainly cytoplasmic (HPA tissue IHC: expression profile). For intact regions, report an H-score from intensity categories 0–3 and their cell percentages, or report the percentage of positive cells. Where cell density differs, add positive cells per mm² and normalize to the number of eligible cells or measured tissue area. Keep retrieval, antibody concentration, DAB development, imaging, and thresholds consistent across the comparison. Present Leydig cells and intestinal glandular cells separately rather than pooling them, since both are reported High but are distinct cell populations (HPA tissue IHC: positive cells).
When is an unexpected ATG4D staining pattern likely to be artefactual?
Check whether staining follows intact cell boundaries and the mainly cytoplasmic tissue pattern before calling it ATG4D positive (HPA tissue IHC: expression profile). Strong signal restricted to section edges, necrotic regions, or the no-primary control warrants investigation of processing or detection background. A predominantly nuclear pattern needs separate support because tissue IHC is mainly cytoplasmic, although nucleoplasmic localisation has cell-imaging support (HPA tissue IHC: expression profile; HPA subcellular: nucleoplasm approved). Compare expected positive Leydig cells with reported negative adipocytes using matched processing where possible (HPA tissue IHC: Leydig cells High; adipocytes Not detected). Exclude residual endogenous peroxidase signal with an appropriate detection control.
Boster reagents
Best ATG4D / Cysteine protease ATG4D IHC Antibodies
ATG4D image data cover human testis IHC and IF (A09793 image captions) and U251 cell IF (M09793 image caption); listed reactivity includes Human, Mouse, and Rat (A09793 catalog).
Real IHC data
Anti-Cysteine protease ATG4D ATG4D Antibody
Cat # A09793
Real IF data
Anti-ATG4D Antibody
Cat # M09793
A09793 lists IHC-P and IF, with human testis images for both applications; its listed reactivity is Human, Mouse, and Rat (A09793 catalog; A09793 image captions). M09793 lists IF and shows U251 cells, including a chloroquine-treated condition; its listed reactivity is Human (M09793 catalog; M09793 IF image caption).
Which to pick: For paraffin-section IHC, choose A09793: IHC-P is listed, and its own image shows human testis staining at 5 μg/mL; the fixative is unreported (A09793 catalog; A09793 IHC image caption). For IF/ICC, M09793 is a mouse monoclonal with a U251 cell IF image at 1:25; A09793 also lists IF and shows human testis IF at 20 μg/mL (M09793 catalog and IF image caption; A09793 catalog and IF image caption). For mouse or rat samples, A09793 is the listed choice because its reactivity includes both species, although its supplied IHC image shows human tissue (A09793 catalog; A09793 IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q86TL0 (ATG4D_HUMAN, Cysteine protease ATG4D).
- Human Protein Atlas. ATG4D tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ATG4D subcellular location (ICC-IF): Localized to the nucleoplasm and mitochondria..
- Human Protein Atlas. ATG4D antibody validation summary (1 antibodies).
- Autophagy Protease ATG4D Facilitates Proliferation and Malignancy of Osteosarcoma Cells. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2025 — PMC12397559.
- Exploring the interplay between EBV and autophagy-related gene expression patterns in nasopharyngeal carcinoma. Frontiers in oncology 2025 — PMC12234470.
- Defective expression of ATG4D abrogates autophagy and promotes growth in human uterine fibroids. Cell death discovery 2017 — PMC5554887.
- A missense change in the ATG4D gene links aberrant autophagy to a neurodegenerative vacuolar storage disease. PLoS genetics 2015 — PMC4398399.
- PubMed PMID:15057824 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:12446702 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ATG4D (Cysteine protease ATG4D). Expected localisation Mainly 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: A09793 at 5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Duodenum (Glandular cells); negative Adipose tissue. 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 A09793); 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-ATG4D, 5 μg/mL (datasheet A09793); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep tissue fixation consistent. (standard IHC practice; not target-specific); Caveat: CASP3 cleavage can shift ATG4D to mitochondria (UniProt); Isoform / epitope: 2 isoforms; cleavage removes residues 1–63, so check epitope (UniProt); Regulation: Expression regulation is unreported (UniProt). Evidence: UniProt Q86TL0, HPA tissue IHC · Approved, HPA ICC-IF · nucleoplasm and mitochondria., Datasheet A09793, PMC12397559, PMC5554887. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATG4D/immunohistochemistry-ihc-guide.