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ATG4B / Cysteine protease ATG4B · IHC design guide
Design Immunohistochemistry for ATG4B
Plan ATG4B IHC-P around cytoplasmic staining in most tissues (HPA tissue IHC). This guide covers consistent section fixation (standard IHC practice), a 5 μg/mL antibody starting concentration (datasheet), and interpretation of nuclear staining flagged as off target (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ATG4B 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 most tissues (HPA tissue IHC) | |
| Staining pattern | Glandular-cell cytoplasmic staining; texture unreported (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Appendix+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 fixation conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Nuclear staining was deemed off target (HPA tissue IHC) | |
| Regulation | Expression regulation unreported (UniProt) | |
| Isoform / epitope | 5 isoforms; epitope coverage unknown (UniProt) |
Section 1
Recommended ATG4B IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with four published ATG4B chromogenic IHC protocols (PMC13313432; PMC9388977; PMC11416411; PMC6966594).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A02885); 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-ATG4B, 5 μg/mL (datasheet A02885) |
| 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 | ATG4B-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in a majority of tissues. 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). Published alternatives include Tris/EDTA pH 9 (PMC11416411; PMC6966594).
Section 2
What Is the Expected ATG4B Staining Pattern?
ATG4B is mainly cytosolic, with smaller pools associated with autophagosomes, endoplasmic reticulum and mitochondria; it has no transmembrane segment (UniProt Q9Y4P1 localization and topology). In tissue IHC, expect cytoplasmic staining in many cell types, especially appendix, colon and duodenal glandular cells (HPA tissue IHC: High). HPA rates its tissue staining Approved, while reporting medium agreement with RNA data and disregarded off-target staining (HPA tissue IHC reliability).
| Cytoplasmic staining in appendix, colon or duodenal glandular cells, with little nuclear signal. | This fits the strongest listed tissue examples: glandular cells are High, and the overall IHC profile is cytoplasmic (HPA tissue IHC). Score the relevant cells and compartment, since a strongly stained area alone does not establish the expected pattern (general IHC practice). |
| Prominent nuclear staining, especially if cytoplasmic staining is weak or absent. | Treat nuclear tissue-IHC signal cautiously: HPA explicitly considered nuclear staining off target and disregarded it (HPA tissue IHC reliability). HPA separately approves nucleoplasmic signal in ICC-IF; that finding does not validate a nuclear pattern on paraffin tissue sections (HPA subcellular ICC-IF; HPA tissue IHC). |
| Strong signal in a cell population listed as Not detected, such as adipocytes or cardiomyocytes. | That conflicts with the listed cell-specific tissue-IHC observations (HPA tissue IHC: adipocytes and cardiomyocytes Not detected). Check cell identification, antibody specificity and detection controls before calling it ATG4B; staining in another cell population within the same tissue needs its own assessment (general IHC practice). |
| Diffuse color covers tissue and empty areas, obscuring cell boundaries. | A non-cellular haze cannot be read as the reported cytoplasmic pattern (HPA tissue IHC profile; general IHC practice). Examine a no-primary control and review blocking, washes and chromogen development to distinguish background from signal in identifiable cells (general IHC practice). |
| No cytoplasmic signal in glandular cells of an otherwise interpretable appendix, colon or duodenum section. | This misses HPA's High cell-specific examples (HPA tissue IHC). Confirm that the intended cells are present and assess the IHC assay with controls before concluding that the specimen lacks ATG4B (general IHC practice). |
💡Expected ATG4B appearanceA convincing positive is discernible cytoplasmic staining in appendix, colon or duodenal glandular cells, where HPA reports High staining; dominant nuclear staining is suspect in tissue IHC (HPA tissue IHC profile, positive cells and reliability).
| Cell type and tissue | HPA reports cytoplasmic expression in a majority of tissues but different cell-specific levels: glandular cells in appendix, colon and duodenum are High, while lung macrophages and pancreatic exocrine glandular cells are Medium (HPA tissue IHC). Compare like cell types when judging intensity. |
| Compartment and topology | The predominant cytosolic location supports a cytoplasmic IHC readout; UniProt also lists autophagosomes, endoplasmic reticulum and mitochondria and reports no transmembrane segment (UniProt Q9Y4P1 localization and topology). These annotations do not require resolvable organelle staining in chromogenic tissue IHC. |
| IHC antibody evidence | The listed IHC antibody CAB037195 is Approved, while HPA reports only medium agreement between tissue staining and RNA and says presumed off-target binding was disregarded (HPA antibody validation; HPA tissue IHC reliability). Interpret unexpected compartments or cells with that qualification in mind. |
| IF/ICC Q: Is nuclear fluorescence an expected tissue-IHC result? | HPA approves nucleoplasm and cytosol in ICC-IF images from MCF-7 and RT-4, but disregards nuclear staining in tissue IHC (HPA subcellular ICC-IF; HPA tissue IHC reliability). Interpret each assay against its own evidence; this section gives no IF/ICC protocol option. |
| Mitochondrial subset | UniProt describes a small mitochondrial portion and reports that Ser-34 phosphorylation promotes mitochondrial localization (UniProt Q9Y4P1 localization). Its presence does not make mitochondrial enrichment a routine tissue-IHC acceptance criterion; HPA's reported tissue pattern remains cytoplasmic (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| High nuclear signal in paraffin tissue sections. | HPA considered nuclear tissue-IHC staining off target, even though nucleoplasmic ICC-IF localization is Approved (HPA tissue IHC reliability; HPA subcellular ICC-IF). | Assess the cytoplasmic pattern separately, include a no-primary control, and avoid scoring nuclei as positive tissue-IHC evidence without independent specificity support (general IHC practice; HPA tissue IHC reliability). |
| No signal in expected positive glandular cells. | Appendix, colon and duodenal glandular cells are High in HPA tissue IHC; an absent result may reflect assay performance or the sampled cell population (HPA tissue IHC; general IHC practice). | Confirm the cells on a counterstained section, then review antigen retrieval, primary-antibody conditions and detection-system controls as general IHC checks; no ATG4B-specific retrieval condition is supplied (general IHC practice). |
| Strong staining in an HPA Not detected cell population. | Unexpected color may represent nonspecific antibody binding or endogenous detection activity; HPA itself notes disregarded presumed off-target staining (general IHC practice; HPA tissue IHC reliability). | Verify cell identity and compare no-primary and detection controls before assigning ATG4B positivity (general IHC practice). |
| Uniform haze or color outside cells. | Background can obscure the cell-bound cytoplasmic profile reported by HPA (general IHC practice; HPA tissue IHC profile). | Check no-primary controls, blocking, washing and chromogen development; judge the result only where cell boundaries and compartment are interpretable (general IHC practice). |
| Weak staining in a tissue expected to be positive. | HPA levels are cell-specific: lung macrophages and pancreatic exocrine glandular cells are Medium, and several other listed cell types are Low (HPA tissue IHC). | Identify the exact cell population and compare it with its HPA level before adjusting assay conditions; use a High glandular-cell example as a positive reference when available (HPA tissue IHC; general IHC practice). |
| Puncta or apparent organelle enrichment dominate the slide. | UniProt lists autophagosome, endoplasmic reticulum and mitochondrial locations, but HPA summarizes tissue IHC as cytoplasmic; organelle identity cannot be assigned from chromogenic appearance alone (UniProt Q9Y4P1 localization; HPA tissue IHC; general IHC practice). | Record the compartment and cell type, check specificity controls, and require independent compartment evidence before naming an organelle (general IHC practice). |
Experiment evidence
IHC & IF images showing ATG4B staining
A02885 · IHCImmunohistochemistry of ATG4B in human spleen tissue with ATG4B antibody at 5 μg/ml.
A02885 · IFImmunofluorescence of ATG4B in human spleen tissue with ATG4B antibody at 20 μg/ml.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000168397-ATG4B/tissue/cerebral+cortex#imid_21016667
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000168397-ATG4B/tissue/colon#imid_21017449
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000168397-ATG4B/tissue/liver#imid_21017450
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000168397-ATG4B/tissue/kidney#imid_21017451
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA069803; green = ATG4B, blue = nucleus, red = microtubules.
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA069803; green = ATG4B, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ATG4B IHC & IF
🧪Run appendix first: glandular cells should stain strongly (HPA: High in appendix glandular cells). Use adipose tissue as the negative comparator (HPA: Not detected in adipocytes); on the appendix slide, treat unstained non-glandular areas as background references only after confirming they are unstained, because HPA does not identify an internal negative cell type.
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ATG4B in MCF-7, RT-4, with annotated localisation: Nucleoplasm (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and immunoglobulin class; use ATG4B knockout material as a biological specificity control (standard IHC practice). For chromogenic appendix IHC, quench endogenous peroxidase and inspect the no-primary slide for tissue background (standard IHC practice).
⚠️Feasibility: A target-specific fixation window, fixation effect, and antigen-retrieval dependency are unreported in the supplied evidence; optimize retrieval on matched sections (standard IHC practice). The selected A02885 spleen IHC caption reports 5 μg/ml but leaves the fixative unreported (selected tissue-IHC caption). The evidence does not establish whether frozen sections or IF are easier; ICC-IF images exist for MCF-7 and RT-4 (HPA subcellular), while appendix may require attention to endogenous peroxidase background in chromogenic IHC (standard IHC practice).
HPA tissue IHC evidence for ATG4B
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded. Nuclear staining was considered off target and disregarded.). 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Lung | Macrophages | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ATG4B IHC Tips
Troubleshoot ATG4B staining in paraffin sections by checking retrieval, cytoplasmic localisation and tissue controls before interpreting chromogenic signal (UniProt Q9Y4P1; HPA tissue IHC).
How should I optimise retrieval when ATG4B staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). If cytoplasmic staining remains weak, compare a more alkaline retrieval buffer on adjacent sections while keeping antibody concentration and detection conditions constant (standard IHC practice). Include a tissue and cell population with reported staining, such as colon glandular cells, to judge whether the change improves the expected pattern (HPA tissue IHC: High in colon glandular cells). Check for tissue lifting and loss of cellular detail after heating, and retain the gentlest condition that gives interpretable cytoplasmic signal (standard IHC practice; HPA tissue IHC: cytoplasmic expression).
Could fixation explain weak or uneven ATG4B staining?
ATG4B-specific sensitivity to fixation is unknown from the supplied evidence; the selected tissue image does not state its fixative (A02885 caption: fixative not stated). Record the fixative, fixation duration and processing history for each paraffin section before comparing staining between samples (standard IHC practice). If fixation varies, test matched sections with the same citrate pH 6.0 retrieval at 95–98 °C for 20 min, then compare morphology and cytoplasmic signal (page retrieval setting; HPA tissue IHC: cytoplasmic expression). Treat differences between batches as a possible processing effect until a consistent cellular pattern and controls support a biological difference (standard IHC practice).
Should nuclear or punctate staining count as ATG4B positive?
Score diffuse cytoplasmic staining as the principal tissue IHC pattern; HPA reports cytoplasmic expression in a majority of tissues (HPA tissue IHC: profile). HPA disregarded nuclear tissue staining as off-target, so exclude nuclear-only chromogenic signal from the ATG4B score (HPA tissue IHC: reliability description). UniProt also lists autophagosomes, endoplasmic reticulum and mitochondria, with a small mitochondrial pool promoted by Ser-34 phosphorylation; those annotations alone do not validate individual puncta in a section (UniProt Q9Y4P1: subcellular location; Ser-34). Compare puncta with the surrounding cytoplasm and a matched negative control before assigning them to a compartment (standard IHC practice).
Can this stain distinguish ATG4B isoforms or phosphorylation states?
Do not assign a chromogenic pattern to one isoform without an epitope map and isoform-specific validation: UniProt lists 5 ATG4B isoforms (UniProt Q9Y4P1: isoforms). The supplied record identifies phosphorylation at Ser-34 and S-nitrosylation at residues 189, 292 and 301, but provides no evidence that the catalog antibody distinguishes those modifications (UniProt Q9Y4P1: modified residues; supplied antibody evidence). ATG4B has no annotated transmembrane segment, so assess accessible cytoplasmic staining without assuming a membrane-restricted epitope (UniProt Q9Y4P1: topology; subcellular location). For a specific isoform or modification claim, obtain the antibody epitope and validate it with an appropriate independent control (standard IHC practice).
How should I adapt the localisation check for multiplex IF?
For the separate IF/ICC workflow, pair ATG4B with a marker that identifies the expected cell population, such as a macrophage marker when examining lung macrophages (HPA tissue IHC: Medium in lung macrophages; standard IF practice). Choose a fluorophore channel after inspecting unstained tissue autofluorescence, and include single-label controls to assess bleed-through (standard IF practice). Because ATG4B is mainly cytosolic and has no transmembrane segment, permeabilise sufficiently for an intracellular epitope while checking that cell boundaries remain intact (UniProt Q9Y4P1: subcellular location; topology; standard IF practice). HPA reports approved nucleoplasmic and cytosolic ICC/IF locations, whereas its tissue IHC assessment disregarded nuclear staining; interpret each application against its own controls (HPA subcellular; HPA tissue IHC: reliability description).
How can I separate ATG4B signal from brown background?
Compare each stained section with a matched section lacking the primary antibody to identify detection-system background (standard chromogenic IHC practice). Apply a peroxidase block before HRP-based DAB detection, and check the control for residual endogenous enzyme activity (standard chromogenic IHC practice). If diffuse brown deposit obscures cell boundaries, reassess antibody concentration, incubation and wash conditions using the same tissue and exposure settings (standard IHC practice). The selected image reports ATG4B staining in human spleen at 5 µg/ml, but its caption does not establish that concentration as optimal for other specimens (A02885 caption). Retain only signal with convincing cytoplasmic localisation and appropriate controls (HPA tissue IHC: profile; standard IHC practice).
What should I measure when ATG4B staining varies across cells? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis area before scoring, then record cytoplasmic intensity and the percentage of positive cells separately (HPA tissue IHC: cytoplasmic profile; standard IHC practice). An H-score can combine intensity categories with their positive-cell percentages; keep thresholds, illumination and DAB development consistent across slides (standard IHC practice). For spatial questions, report positive cells per mm² of viable tissue and normalise to the number of eligible cells or viable area, as appropriate (standard IHC practice). Exclude nuclear-only staining from the ATG4B score because HPA disregarded it in tissue IHC, and report the exclusion rule (HPA tissue IHC: reliability description).
When is an apparent ATG4B-positive area likely to be artefact?
Favour cytoplasmic signal in intact cells over nuclear-only staining: HPA reports broad cytoplasmic tissue expression and disregarded nuclear tissue staining as off-target (HPA tissue IHC: profile; reliability description). Check cell identity against the expected compartment; for example, colon glandular cells are reported High, while adipocytes are reported Not detected (HPA tissue IHC: colon; adipose tissue). Treat staining confined to section edges, folds or necrotic areas as suspect, and compare those areas with intact tissue on the same slide (standard IHC practice). If brown signal persists without primary antibody, investigate endogenous peroxidase or detection background before interpreting it as ATG4B (standard chromogenic IHC practice).
Boster reagents
Best ATG4B / Cysteine protease ATG4B IHC Antibodies
ATG4B antibody A02885 has human spleen IHC and IF images (catalog image captions); its listed reactivity covers human, mouse, and rat (catalog: reactivity).
Real IHC data
Anti-Cysteine protease ATG4B ATG4B Antibody
Cat # A02885
A02885 has an IHC image of human spleen tissue at 5 μg/mL (catalog: IHC image caption). The same SKU has an IF image of human spleen tissue at 20 μg/mL (catalog: IF image caption).
Which to pick: Choose A02885 for paraffin section IHC (catalog: IHC-P application); its IHC image shows human spleen tissue, but the caption does not report the fixative (catalog: IHC image caption). Choose A02885 for IF (catalog: IF application and IF image caption); ICC is not listed as a tested application (catalog: applications). For mouse or rat work, A02885 lists both species as reactive, although its supplied IHC and IF images show human tissue; the host is rabbit and clonality is unreported (catalog: reactivity, image captions, host, clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y4P1 (ATG4B_HUMAN, Cysteine protease ATG4B).
- Human Protein Atlas. ATG4B tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ATG4B subcellular location (ICC-IF): Localized to the nucleoplasm and cytosol..
- Human Protein Atlas. ATG4B antibody validation summary (2 antibodies).
- Upregulated ATG4B predicts poor prognosis and correlates with angiogenesis in osteosarcoma. Journal of the Egyptian National Cancer Institute 2025 — PMC13313432.
- Nitidine chloride induces cardiac hypertrophy in mice by targeting autophagy-related 4B cysteine peptidase. Acta pharmacologica Sinica 2023 — PMC9388977.
- TDP-43 regulates LC3ylation in neural tissue through ATG4B cryptic splicing inhibition. Acta neuropathologica 2024 — PMC11416411.
- Association of ATG4B and Phosphorylated ATG4B Proteins with Tumorigenesis and Prognosis in Oral Squamous Cell Carcinoma. Cancers 2019 — PMC6966594.
- PubMed PMID:12446702 — UniProt-cited evidence.
- PubMed PMID:30808002 — UniProt-cited evidence.
- PubMed PMID:15169837 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ATG4B (Cysteine protease ATG4B). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A02885 at 5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (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 A02885); 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-ATG4B, 5 μg/mL (datasheet A02885); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Nuclear staining was deemed off target (HPA tissue IHC); Isoform / epitope: 5 isoforms; epitope coverage unknown (UniProt); Regulation: Expression regulation unreported (UniProt). Evidence: UniProt Q9Y4P1, HPA tissue IHC · Approved, HPA ICC-IF · nucleoplasm and cytosol., Datasheet A02885, PMC13313432, PMC9388977. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATG4B/immunohistochemistry-ihc-guide.