This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
ATG4D / Cysteine protease ATG4D · IHC design guide
Design Immunohistochemistry for ATG4D
Plan chromogenic IHC on paraffin sections with the IHC-validated antibody at a starting concentration of 5 μg/mL (datasheet: A09793). Use duodenal glandular cells or testicular Leydig cells as positive tissue controls, and assess mainly cytoplasmic staining (HPA tissue IHC).
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 | Tissue: mainly cytoplasmic (HPA tissue IHC); mitochondrial matrix after CASP3 cleavage (UniProt) | |
| Staining pattern | Mainly cytoplasmic in glandular and Leydig cells (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 | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A09793) | |
| Caveat | Staining has medium consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Expression regulation is unreported (UniProt) | |
| Isoform / epitope | 2 isoforms; CASP3 cleavage removes residues 1–63, so epitope position matters (UniProt) |
Section 1
Recommended ATG4D IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with four published ATG4D tissue IHC methods (PMC12397559; PMC12234470; 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 rule); the published methods give alternatives where specified.
Section 2
What Is the Expected ATG4D Staining Pattern?
In paraffin-section IHC, expect mainly cytoplasmic ATG4D staining, especially in duodenal and small-intestinal glandular cells and testicular Leydig cells (HPA tissue IHC: High; profile: mainly cytoplasmic). ATG4D has no transmembrane segment, and UniProt also places it in the mitochondrial matrix after CASP3 cleavage during oxidative stress or cell death (UniProt Q86TL0 topology and subcellular location). HPA rates its tissue IHC evidence Approved, with medium consistency against RNA expression (HPA tissue IHC).
| Cytoplasmic staining is strong in duodenal or small-intestinal glandular cells, or testicular Leydig cells. | This matches the expected cell types, compartment and High staining level (HPA tissue IHC). Compare signal with neighboring cells and a negative control on the same run (general IHC practice). |
| Staining is predominantly nuclear, with little cytoplasmic signal in a paraffin section. | Recheck specificity and detection conditions: the HPA tissue IHC profile is mainly cytoplasmic (HPA tissue IHC). Nuclear signal alone is not proof of artefact, because HPA reports nucleoplasmic localisation by ICC-IF (HPA subcellular). |
| Strong staining appears in adipocytes, cardiomyocytes or skeletal myocytes. | These cell types are reported as Not detected in HPA tissue IHC (HPA tissue IHC). Consider cross-reactivity or endogenous detection activity, and compare with a no-primary control (general IHC practice). |
| Brown signal is diffuse across cells, stroma or the entire section without a cell-specific pattern. | This does not reproduce the mainly cytoplasmic, cell-type-dependent tissue profile (HPA tissue IHC). Review blocking, washes and detection background with a no-primary control (general IHC practice). |
| No staining appears in duodenal or small-intestinal glandular cells or testicular Leydig cells. | These are High-staining reference cell types, so an absent signal calls for a run check (HPA tissue IHC). Review section integrity, antigen retrieval and detection controls before interpreting the specimen as negative (general IHC practice). |
💡Expected ATG4D appearanceA convincing IHC-positive result is High, mainly cytoplasmic staining in intestinal glandular cells or testicular Leydig cells (HPA tissue IHC); uniform staining of cells HPA calls Not detected, or section-wide background, warrants a specificity check (HPA tissue IHC; general IHC practice).
| Compartment and processing | UniProt places full-length ATG4D in the cytoplasm and reports mitochondrial-matrix import after CASP3 cleavage during oxidative stress or cell death (UniProt Q86TL0). A mitochondrial pattern should therefore be interpreted in context; the HPA paraffin-section profile remains mainly cytoplasmic (HPA tissue IHC). |
| Sequence and isoforms | ATG4D has no annotated transmembrane segment and has two isoforms (UniProt Q86TL0). UniProt lists full-length residues 1–474 and a mitochondrial chain spanning residues 64–474 (UniProt Q86TL0); the supplied evidence does not identify the antibody epitope or establish which forms it detects. |
| Tissue controls and validation | Duodenal and small-intestinal glandular cells and testicular Leydig cells provide High-staining comparisons; adipocytes, cardiomyocytes and skeletal myocytes are reported as Not detected (HPA tissue IHC). The tissue IHC reliability is Approved with medium RNA-to-staining consistency, and HPA067683 is IHC Approved (HPA tissue IHC; HPA antibody validation). |
| IF/ICC interpretation | What if IF shows nuclear or mitochondrial signal? HPA reports nucleoplasm as approved and mitochondria as supported in ICC-IF, with U2OS images; HPA067683 is ICC Supported (HPA subcellular; HPA antibody validation). Those ICC-IF observations do not replace the mainly cytoplasmic paraffin-section IHC expectation (HPA tissue IHC). |
| Retrieval and fixation evidence | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive glandular or Leydig cells show no chromogenic signal. | The run may have failed, or retrieval or detection may be inadequate (general IHC practice); these cells are High in HPA tissue IHC (HPA tissue IHC). | Check the positive section and detection controls, then optimize retrieval within the assay workflow (general IHC practice). Do not score the test section as ATG4D-negative until controls work. |
| The whole section develops a similar brown haze. | Non-specific background or endogenous detection activity can obscure cell-specific staining (general IHC practice); HPA describes a mainly cytoplasmic tissue pattern (HPA tissue IHC). | Run a no-primary control; review blocking, wash stringency and chromogen development, then reassess whether the expected cell types stand out (general IHC practice; HPA tissue IHC). |
| Adipocytes, cardiomyocytes or myocytes stain as strongly as expected positive cells. | Cross-reactivity or endogenous detection activity is possible (general IHC practice); HPA reports these cell types as Not detected (HPA tissue IHC). | Compare a no-primary control and the HPA High-staining reference cells in the same run; interpret unexpected positivity cautiously (general IHC practice; HPA tissue IHC). |
| The signal is chiefly nuclear in paraffin-section IHC. | This differs from the mainly cytoplasmic tissue profile, although nucleoplasmic localisation is reported in ICC-IF (HPA tissue IHC; HPA subcellular). | Check the negative control and compare cytoplasmic staining in HPA High-staining cell types before assigning the nuclear signal to ATG4D (general IHC practice; HPA tissue IHC). |
| Punctate signal is interpreted as mitochondrial ATG4D. | Mitochondrial-matrix import is reported after CASP3 cleavage during oxidative stress or cell death, and mitochondrial localisation is supported in ICC-IF (UniProt Q86TL0; HPA subcellular). IHC puncta alone do not establish organelle identity. | Report the observed pattern as punctate unless an appropriate colocalisation experiment establishes mitochondrial localisation (general IF practice). Keep the tissue IHC expectation anchored to HPA’s mainly cytoplasmic profile (HPA tissue IHC). |
| A weakly stained test section is called negative after comparison with another tissue. | HPA reports Medium staining in several cell types and Low staining in others, so expected intensity depends on the sampled cell type (HPA tissue IHC). | Identify and score the relevant cells, compare an appropriate HPA High-staining reference, and verify run controls before concluding that weak signal is absent (HPA tissue IHC; 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: Leydig cells should stain (HPA: High in testis Leydig cells; selected A09793 tissue-IHC caption: testis staining). Run skeletal muscle as a negative tissue: myocytes are listed as Not detected (HPA: skeletal muscle myocytes); on the testis slide, score any unstained neighboring cells as internal background without assuming every non-Leydig cell is negative.
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 a no-primary, secondary-only control; use host-matched nonimmune IgG for a polyclonal primary or a host- and isotype-matched control for a monoclonal primary; and confirm specificity with ATG4D knockout tissue or a validated peptide block (standard IHC practice). Block endogenous peroxidase and inspect the testis control for residual chromogenic background (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A09793 testis IHC caption does not state a fixative (selected A09793 tissue-IHC caption: fixative unreported). Antigen retrieval dependence is unreported, so optimize retrieval on the positive control (standard IHC practice). The supplied evidence does not establish whether frozen sections or IF are easier; U2OS ICC-IF images exist (HPA: U2OS ICC-IF), while no testis-specific artefact is reported in the supplied evidence.
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
Troubleshoot ATG4D staining in paraffin sections by checking retrieval, tissue context, compartment, controls, and scoring before interpreting chromogenic signal.
How should I retrieve ATG4D in paraffin sections when staining is weak?
Use citrate buffer at pH 6.0 for heat-induced retrieval at 95–98 °C for 20 min (page retrieval rule). Let sections cool consistently, then compare the same tissue and antibody dilution across retrieval runs so that heating remains the main variable (standard IHC practice). If staining is weak, test a different retrieval buffer or pH on adjacent sections as a fallback, with a matched control in each run (standard IHC practice). Examine morphology alongside signal: excessive heating can damage sections and make apparent cytoplasmic staining difficult to assign to cells (standard IHC practice).
Could fixation explain weak or uneven ATG4D staining?
Target-specific fixation sensitivity is unknown from the supplied evidence; the tissue-IHC caption reports 5 µg/ml antibody in human testis but does not state a fixative (caption A09793). Record the fixative, fixation duration, and section processing for every specimen, then compare sections processed alike before changing antibody concentration (standard IHC practice). If staining varies between blocks, assess tissue preservation and retrieval performance with appropriate control sections from the same run (standard IHC practice). Avoid attributing a fixation effect to ATG4D solely from its reported localisation or modification, because those annotations do not establish fixation sensitivity (UniProt Q86TL0).
Where should ATG4D signal appear in tissue sections?
Expect predominantly cytoplasmic tissue staining as a starting pattern (HPA tissue IHC: mainly cytoplasmic expression). ATG4D is annotated in the cytoplasm and mitochondrial matrix, with mitochondrial import after CASP3 cleavage during oxidative stress and cell death (UniProt Q86TL0). Nucleoplasmic and mitochondrial localisation is also reported from cell imaging, so assess a nuclear pattern against tissue controls before accepting it in chromogenic sections (HPA subcellular). Score the compartment within identified cells, not diffuse colour across a field, and compare serial sections when morphology is ambiguous (standard IHC practice). A mitochondrial contribution may appear as cytoplasmic staining at routine light-microscope resolution (standard IHC interpretation).
Can this stain distinguish ATG4D isoforms or the mitochondrial form?
Do not assign an isoform from staining alone: 2 ATG4D isoforms are listed, but the supplied antibody caption does not map an epitope (UniProt Q86TL0; caption A09793). The annotated full-length chain spans residues 1–474, whereas the mitochondrial chain spans 64–474 after cleavage (UniProt Q86TL0). An N-terminal epitope might be absent from the processed chain, but that possibility cannot be evaluated without an epitope map (UniProt Q86TL0; standard immunostaining interpretation). Phosphoserine at residue 467 is annotated, yet staining cannot establish phosphorylation state without a validated modification-specific reagent (UniProt Q86TL0; standard IHC practice).
How should I follow up an ATG4D IHC pattern with multiplex IF?
Choose an independently validated marker for the cell population under study, then confirm whether ATG4D and that marker occupy the same cells (standard IF practice). For a tissue comparison, Leydig cells in testis and glandular cells in duodenum or small intestine are reported with high staining (HPA tissue IHC). Select fluorophores after checking unstained tissue autofluorescence, and favor a spectrally separated channel with adequate signal over a weak channel dominated by background (standard IF practice). Because ATG4D has no annotated transmembrane segment, permeabilise to access intracellular epitopes, while adjusting detergent conditions to the antibody’s mapped epitope and desired compartment (UniProt Q86TL0; standard IF practice).
How can I reduce diffuse brown staining without losing ATG4D signal?
First compare a no-primary control with the ATG4D section to identify signal from the detection system or tissue itself (standard IHC practice). Block endogenous peroxidase before chromogenic detection, and use an appropriate protein block and thorough washes to reduce nonspecific binding (standard IHC practice). Titrate the antibody around the reported 5 µg/ml tissue-IHC use, while recognizing that its caption does not specify fixation or paraffin processing (caption A09793). Judge each change in a matched positive area and a lower-signal area; adipocytes are reported as not detected and testis Leydig cells as high (HPA tissue IHC).
How should I quantify ATG4D staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell type and compartment before scoring, because tissue IHC reports mainly cytoplasmic expression and different levels across cell populations (HPA tissue IHC). For a consistent chromogenic assay, record the percentage of positive cells and intensity grades, then calculate an H-score from 0–300 if those grades are reproducible (standard IHC practice). Alternatively, report positive-cell density per mm² when cell counts per area better fit the specimen (standard IHC practice). Normalise comparisons to the number of eligible cells or measured tissue area, and hold staining, imaging, thresholding, and region selection constant across samples (standard IHC practice).
When is an ATG4D-positive area credible rather than artefactual?
A credible result has cellular staining in an expected compartment and a pattern that recurs in comparable sections (standard IHC interpretation). Testis Leydig cells, duodenal glandular cells, and small-intestinal glandular cells are reported with high staining, whereas adipocytes and cardiomyocytes are reported as not detected (HPA tissue IHC). Treat isolated nuclear-only colour cautiously against the mainly cytoplasmic tissue profile, while remembering that nucleoplasmic localisation has been reported in cell imaging (HPA tissue IHC; HPA subcellular). Exclude section-edge enhancement, necrotic deposits, and residual endogenous enzyme signal with morphology and no-primary controls before calling a region positive (standard IHC practice).
Boster reagents
Best ATG4D / Cysteine protease ATG4D IHC Antibodies
ATG4D antibodies have tissue IHC data in human testis (A09793 image caption) and cell IF data in U251 cells (M09793 image caption); catalog 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 will render with human testis IHC at 5 μg/mL (A09793 IHC image caption); its catalog lists IHC-P and IF applications (A09793 catalog). M09793 will render with U251 cell IF at 1:25 (M09793 IF image caption); its catalog lists IF, but not IHC-P (M09793 catalog).
Which to pick: Choose A09793 for paraffin-section IHC: it lists IHC-P (A09793 catalog) and has a human testis IHC image at 5 μg/mL (A09793 IHC image caption); the caption does not report the fixative (A09793 IHC image caption). For cell IF, choose monoclonal M09793, shown in U251 cells (M09793 catalog; M09793 IF image caption). For mouse or rat work, A09793 is the listed cross-species option (A09793 catalog reactivity), although its supplied IHC image shows human tissue (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 Tissue: mainly cytoplasmic (HPA tissue IHC); mitochondrial matrix after CASP3 cleavage (UniProt). 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: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A09793); Caveat: Staining has medium consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 2 isoforms; CASP3 cleavage removes residues 1–63 , so epitope position matters (UniProt); Regulation: Expression regulation is unreported (UniProt). Evidence: UniProt Q86TL0, HPA tissue IHC · Approved, HPA ICC-IF · nucleoplasm and mitochondria., Datasheet A09793, PMC12397559, PMC12234470. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATG4D/immunohistochemistry-ihc-guide.