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ATF5 / Cyclic AMP-dependent transcription factor ATF-5 · IHC design guide
Design Immunohistochemistry for ATF5
Plan ATF5 chromogenic IHC on paraffin sections with a 1:50 catalog antibody starting dilution (datasheet M02792). Assess nuclear staining with caution because tissue IHC and RNA expression show low consistency (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ATF5 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 | Nuclear staining in tissue (HPA tissue IHC); cytoplasm and centrosome annotated (UniProt) | |
| Staining pattern | General nuclear staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M02792) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent between sections (standard IHC practice; not target-specific) | |
| Caveat | Low IHC–RNA concordance limits tissue predictions (HPA tissue IHC) | |
| Regulation | NPM1 promotes ATF5 degradation (UniProt) | |
| Isoform / epitope | No listed isoforms; one 1–282 chain (UniProt) |
Section 1
Recommended ATF5 IHC & IF Protocols
The catalog antibody’s IHC protocol is paired with published paraffin-section methods for thyroid, rat heart, and human nucleus pulposus tissue (PMC5067000; PMC8677764; PMC13202852).
| Sample | Paraffin-embedded human pancreas cancer tissue; fixative not specified (datasheet M02792) |
| Fixation | Image fixative and duration unreported (datasheet M02792); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet M02792); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M02792) |
| Primary antibody | Rabbit monoclonal (clone HDD-1) anti-ATF5, 1:50 recommended; image 1:100 (datasheet M02792) |
| Primary incubation | Overnight at 4 °C (datasheet M02792) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M02792) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ATF5-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet M02792); the rat heart and human nucleus pulposus articles used citrate pH 6.0 (PMC8677764; PMC13202852).
Section 2
What Is the Expected ATF5 Staining Pattern?
ATF5 is mainly nuclear, with cytoplasmic and centrosomal localization also reported (UniProt Q9Y2D1; HPA: general nuclear expression). In paraffin tissue IHC, expect staining in the cell types HPA reports as positive, including adrenal glandular cells, bronchial respiratory epithelial cells and cerebellar Purkinje cells (HPA: High). Interpret intensity cautiously: HPA rates its tissue IHC profile Uncertain because antibody staining and RNA expression have low consistency (HPA: Uncertain). ATF5 has no transmembrane segment (UniProt Q9Y2D1 topology).
| Nuclear staining in adrenal glandular cells or cerebellar Purkinje cells (HPA: High). | This fits the reported tissue pattern and ATF5 localization (HPA: High in these cells; UniProt Q9Y2D1: nucleus). Judge the result against the cell type, not overall tissue darkness (standard IHC practice). The match is supportive, but HPA's tissue IHC reliability remains Uncertain (HPA: Uncertain). |
| Cytoplasmic signal accompanies nuclear staining, or a small pericentrosomal focus is visible. | These locations can be compatible with ATF5 (UniProt Q9Y2D1: cytoplasm and centrosome). A focus alone does not identify ATF5: the reported centrosomal pattern is cell cycle dependent, and routine chromogenic IHC may not resolve it (UniProt Q9Y2D1; standard IHC practice). |
| Predominantly membrane, extracellular or uniformly cytoplasmic staining, without a convincing nuclear pattern. | Treat this as a localization mismatch and investigate an artefact before calling it ATF5 (HPA: general nuclear expression; UniProt Q9Y2D1: no transmembrane segment). Cytoplasmic ATF5 is possible, so diffuse cytoplasm alone cannot prove cross-reactivity (UniProt Q9Y2D1: cytoplasm). |
| Strong staining in adipocytes, especially if reported positive cell types lack nuclear signal. | That conflicts with HPA's adipocyte result and warrants checks for cross-reactivity or endogenous detection activity (HPA: Not detected in adipocytes; standard IHC practice). It is a warning pattern, not proof of either cause; HPA rates the tissue IHC profile Uncertain (HPA: Uncertain). |
| Diffuse slide-wide color or no signal in a reported high-staining cell population. | Diffuse color obscures cell and compartment scoring; absent signal leaves the run uninterpretable until controls are checked (standard IHC practice). Compare with a reported high-staining population, such as bronchial respiratory epithelial cells (HPA: High), while retaining the HPA reliability caveat (HPA: Uncertain). |
💡Expected ATF5 appearanceA plausible positive is distinct, predominantly nuclear staining in a reported High cell population, such as adrenal glandular cells (HPA: High; HPA: general nuclear expression); widespread cell-independent color or membrane-only staining is suspect (standard IHC practice; UniProt Q9Y2D1 topology).
| Tissue and cell selection | HPA reports High staining in several epithelial and glandular populations, Purkinje cells and glomerular cells, but Not detected in adipocytes (HPA: tissue IHC). Select controls by the annotated cell population; tissue names alone can conceal mixed results (standard IHC practice). |
| Liver RNA versus IHC | ATF5 is widely expressed with higher expression in liver, and HPA calls liver RNA tissue enriched (UniProt Q9Y2D1; HPA: RNA specificity). Yet HPA reports Low staining in hepatocytes (HPA: Low). Do not use liver RNA enrichment to predict strong hepatocyte IHC signal. |
| Antibody evidence | HPA030187 is rated IHC Uncertain and ICC Supported (HPA: antibody validation). HPA also rates the tissue IHC profile Uncertain for low consistency with RNA data (HPA: reliability). These ratings limit confidence in a single staining pattern; they do not establish a validated IHC protocol. |
| Subcellular interpretation | ATF5 is listed in nucleus, cytoplasm and centrosome, with movement to the midbody during late division (UniProt Q9Y2D1). HPA ICC-IF mainly places it in nucleoplasm, with vesicles and cytosol also reported (HPA: subcellular). These observations allow more than one intracellular pattern. |
| IF/ICC Q&A: What localization should I expect? | Mainly nucleoplasmic signal, with possible vesicular and cytosolic signal (HPA: ICC-IF subcellular; HPA030187 ICC Supported). This is interpretation guidance for IF/ICC; the application has its own guide page. |
| Situation | Likely cause | Next action |
|---|---|---|
| No nuclear signal in a reported High population (HPA: tissue IHC). | The run or detection may have failed; a single negative section cannot establish absent ATF5 (standard IHC practice; HPA: Uncertain). | Check the positive control, primary-antibody step and detection reagents, then repeat before scoring the tissue negative (standard IHC practice). |
| Strong diffuse brown color across cells and stroma. | Background from detection chemistry or insufficient blocking may obscure localization (standard chromogenic IHC practice). | Compare a no-primary control; review blocking, washes and chromogen development, then score only cell-resolved signal (standard IHC practice). |
| Strong staining in adipocytes (HPA: Not detected). | Cross-reactivity or endogenous detection activity is possible; the pattern alone cannot distinguish them (standard IHC practice; HPA: Uncertain). | Compare a no-primary control and a reported High cell population in the same run before attributing adipocyte color to ATF5 (standard IHC practice; HPA: tissue IHC). |
| Signal is membrane-only or extracellular. | The compartment conflicts with ATF5's reported intracellular localization and lack of a transmembrane segment (UniProt Q9Y2D1 topology and subcellular location). | Inspect the no-primary control and tissue morphology; seek a reproducible nuclear pattern in a reported positive cell type (standard IHC practice; HPA: general nuclear expression). |
| Hepatocytes stain weakly despite liver RNA enrichment. | HPA reports Low hepatocyte IHC alongside liver-enriched RNA; its tissue IHC reliability is Uncertain (HPA: Low in hepatocytes; RNA tissue enriched in liver; Uncertain). | Do not raise the expected IHC intensity from RNA alone. Check a reported High population and score hepatocyte staining on its own merits (HPA: tissue IHC; standard IHC practice). |
| Only tiny perinuclear dots are visible. | ATF5 can localize to centrosomes, but chromogenic dots alone do not verify their identity (UniProt Q9Y2D1: centrosome; standard IHC practice). | Record the pattern without calling each dot ATF5; compare controls and assess whether the expected nuclear signal is present (standard IHC practice; HPA: general nuclear expression). |
Experiment evidence
IHC & IF images showing ATF5 staining
M02792 · IHCIHC analysis of ATF5 using anti-ATF5 antibody (M02792). ATF5 was detected in a paraffin-embedded section of human pancreas cancer tissue. He
M02792 · IHCIHC analysis of ATF5 using anti-ATF5 antibody (M02792). ATF5 was detected in a paraffin-embedded section of mouse liver tissue. Heat mediate
M02792 · IFICC staining of ATF5 in Hela cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room tem
A02792 · IFICC staining ATF5 in Hela cells (green). The nuclear counter stain is DAPI (blue). Cells were fixed in paraformaldehyde, permeabilised with
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000169136-ATF5/tissue/cerebral+cortex#imid_7458816
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000169136-ATF5/tissue/colon#imid_7458678
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000169136-ATF5/tissue/liver#imid_7458671
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000169136-ATF5/tissue/kidney#imid_7458672
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA030187; green = ATF5, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA030187; green = ATF5, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ATF5 IHC & IF
🧪Run adrenal gland first: glandular cells should stain (HPA: High in adrenal gland glandular cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the adrenal slide, cells without specific signal should show only background staining, without a defined ATF5 pattern (standard IHC practice).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ATF5 in A-431, U-251MG, U2OS, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a matched rabbit IgG isotype control, and an ATF5-knockout specimen if available (caption: rabbit primary antibody; standard IHC practice). Quench endogenous peroxidase for DAB detection and use the no-primary slide to distinguish any adrenal pigment from chromogen (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected M02792 paraffin-section caption does not state a fixative (caption: fixative unreported). That caption documents heat retrieval in EDTA at pH 8.0, but does not establish whether retrieval is required across tissues (caption: EDTA retrieval). Neither frozen sections nor IF can be judged easier from the supplied evidence; HPA ICC-IF supports mainly nucleoplasmic localization, with additional cytosolic and vesicular signal, while endogenous adrenal pigment may complicate interpretation of brown DAB staining (HPA: subcellular localization; standard IHC practice).
HPA tissue IHC evidence for ATF5
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data. 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ATF5 IHC Tips
Use the catalog antibody’s paraffin-section result as a starting point, and interpret ATF5 staining in light of its reported compartments and uncertain tissue-IHC reliability (datasheet M02792; UniProt Q9Y2D1; HPA tissue IHC).
How should I adjust retrieval when ATF5 staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet M02792). The selected tissue-IHC image used this condition before overnight incubation with antibody at 1:100 and 4°C (datasheet M02792). If staining remains weak, compare a modest range of heating times on matched sections while keeping cooling and detection conditions constant (standard IHC practice). Excessive retrieval can damage tissue morphology or increase background, so judge each condition against a no-primary control and preserved nuclear detail (standard IHC practice). A citrate buffer trial should be treated as a fallback experiment, since the selected image documents EDTA rather than citrate (datasheet M02792).
Could fixation explain weak or uneven ATF5 staining?
The selected paraffin-section caption does not state a fixative, so ATF5-specific fixation sensitivity is unknown for this antibody (datasheet M02792). Record the fixative, fixation duration, tissue thickness, and time before fixation for each specimen before comparing staining intensities (standard IHC practice). Compare matched sections processed together, and keep retrieval at EDTA pH 8.0 while assessing whether fixation history tracks the signal (datasheet M02792; standard IHC practice). If fixation varies across specimens, avoid interpreting a difference in DAB intensity as a biological difference without a processing-matched control (standard IHC practice). HPA staining patterns do not establish how this antibody responds to fixation (HPA tissue IHC).
Should ATF5 staining be nuclear, cytoplasmic, or centrosomal?
Assess nuclear staining first: HPA describes general nuclear tissue expression and a supported nucleoplasmic location in cell images (HPA tissue IHC; HPA subcellular). Cytoplasmic staining can also be plausible because ATF5 is reported in the cytoplasm, and HPA reports cytosol and vesicles as additional locations (UniProt Q9Y2D1; HPA subcellular). UniProt also describes centrosomal accumulation from G1 through M phase and movement to the midbody during late telophase and cytokinesis (UniProt Q9Y2D1). Resolve tiny puncta only when section quality and optical resolution permit; diffuse DAB deposits alone cannot establish centrosomal localisation (standard IHC practice). Score nuclear, cytoplasmic, and punctate patterns separately, with no-primary controls beside each comparison (standard IHC practice).
What should I check if this antibody gives an unexpected staining pattern?
The supplied record lists 0 isoforms, one ATF5 chain spanning residues 1–282, and a bZIP domain at 208–271 (UniProt Q9Y2D1). It also lists acetylation at residue 29 and phosphorylation at residue 256, but supplies no epitope location for this antibody (UniProt Q9Y2D1; datasheet M02792). Do not attribute altered staining to an isoform or modification without direct epitope and modification evidence (UniProt Q9Y2D1; standard IHC practice). First compare retrieval conditions, antibody concentration, and no-primary controls on adjacent sections, then seek an independently validated reagent if specificity remains uncertain (datasheet M02792; standard IHC practice).
How can I use IF to check which cells contain ATF5?
Use IF as a separate validation experiment, since the selected antibody evidence here describes chromogenic staining of a paraffin section (datasheet M02792). Co-stain with a validated marker for the cell population under study and compare ATF5 signal within marker-positive and marker-negative cells (standard IF practice). Choose fluorophores after inspecting unstained tissue autofluorescence, favoring a spectrally separated channel with low background for the weaker signal (standard IF practice). ATF5 has no transmembrane segment and is reported in nuclear and cytoplasmic compartments, so use controlled permeabilisation to give antibody access to those intracellular epitopes (UniProt Q9Y2D1; standard IF practice). Include single-stain and secondary-only controls when evaluating colocalisation and bleed-through (standard IF practice).
How do I reduce diffuse or granular DAB background?
Compare the complete stain with a no-primary section to separate antibody-dependent signal from detection-system deposits (standard IHC practice). The selected image used 10% goat serum blocking, antibody at 1:100 overnight at 4°C, and DAB development (datasheet M02792). Keep washes consistent, check whether shorter DAB development preserves specific nuclear contrast, and titrate the primary around the documented concentration on matched sections (datasheet M02792; standard IHC practice). Include a peroxidase-blocking step and inspect a no-primary control for residual endogenous enzyme activity; these are general chromogenic IHC safeguards (standard IHC practice). Treat granular pigment or staining concentrated at section folds as suspect until controls resolve it (standard IHC practice).
What is a defensible way to quantify ATF5 IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue compartment and eligible cell population before scoring, then count nuclei with ATF5 staining separately from cytoplasmic signal (HPA tissue IHC; UniProt Q9Y2D1; standard IHC practice). Report the percentage of positive cells and, if intensity categories are reproducible, an H-score using 0–3 intensity bins and the percentage in each bin (standard IHC practice). Normalise counts to the number of eligible cells, or report positive-cell density per mm² of viable tissue when cellularity differs (standard IHC practice). Apply one threshold and imaging setup across matched sections, excluding folds, necrosis, and edge artefacts (standard IHC practice). State the compartment and denominator because HPA tissue-IHC reliability for ATF5 is uncertain (HPA tissue IHC).
How can I distinguish genuine ATF5 staining from artefact?
Give greatest weight to reproducible nuclear staining in intact cells, while allowing biologically plausible cytoplasmic signal because both compartments are reported for ATF5 (HPA tissue IHC; UniProt Q9Y2D1). Check whether the stained cells match the intended cell population; HPA reports high staining in several specified populations, but rates its tissue-IHC evidence uncertain (HPA tissue IHC). Distrust signal confined to section edges, folds, or necrotic areas, and compare it with a no-primary section to identify nonspecific deposits (standard IHC practice). Residual endogenous peroxidase can produce DAB signal, so confirm peroxidase blocking and review the control before calling diffuse brown staining positive (standard IHC practice). Corroborate consequential findings with an independent assay (standard IHC practice).
Boster reagents
Best ATF5 / Cyclic AMP-dependent transcription factor ATF-5 IHC Antibodies
Anti-ATF5 IHC images cover paraffin sections of human pancreas cancer and mouse liver (M02792 image captions); an ICC/IF image covers HeLa cells (A02792 image caption). Both antibodies list human, mouse and rat reactivity (catalog).
Real IHC data
Anti-ATF5 Rabbit Monoclonal Antibody
Cat # M02792
Real IF data
Anti-ATF5 Antibody
Cat # A02792
M02792 will render with its IHC figure from a human pancreas cancer paraffin section; a second caption documents mouse liver (M02792 image captions). A02792 will render with its ICC/IF figure from HeLa cells (A02792 image caption).
Which to pick: For tissue IHC, choose M02792: its own captions document staining in human pancreas cancer and mouse liver paraffin sections, but do not report the fixative (M02792 image captions). For IF/ICC, choose A02792, whose own figure documents paraformaldehyde-fixed, permeabilised HeLa cells (A02792 image caption). For cross-species studies, both list human, mouse and rat reactivity (catalog: M02792, A02792); M02792 is rabbit monoclonal clone HDD-1 (catalog: M02792), while A02792 lists IHC and IF/ICC applications without a specified clone (catalog: A02792).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y2D1 (ATF5_HUMAN, Cyclic AMP-dependent transcription factor ATF-5).
- Human Protein Atlas. ATF5 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. ATF5 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to vesicles and cytosol..
- Human Protein Atlas. ATF5 antibody validation summary (1 antibodies).
- Differential expression of two activating transcription factor 5 isoforms in papillary thyroid carcinoma. OncoTargets and therapy 2016 — PMC5067000.
- Altered cardiac mitochondrial dynamics and biogenesis in rat after short-term cocaine administration. Scientific reports 2021 — PMC8677764.
- Expression and targeting of transcription factor ATF5 in dog gliomas. Veterinary and comparative oncology 2018 — PMC5677578.
- From collapse to comeback: Luteolin rejuvenates nucleus pulposus progenitor cells via SIRT1/ATF5-UPRmt to reverse intervertebral disc degeneration cascade. Stem cell research & therapy 2026 — PMC13202852.
- PubMed PMID:11087824 — UniProt-cited evidence.
- PubMed PMID:15221005 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ATF5 (Cyclic AMP-dependent transcription factor ATF-5). Expected localisation Nuclear staining in tissue (HPA tissue IHC); cytoplasm and centrosome annotated (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M02792). Validated IHC antibody: M02792 at 1:50 recommended; image 1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human pancreas cancer tissue; fixative not specified (datasheet M02792); 2. Fixation: Image fixative and duration unreported (datasheet M02792); 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 retrieval: EDTA pH 8.0 (datasheet M02792); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M02792); 8. Primary antibody: Rabbit monoclonal (clone HDD-1) anti-ATF5, 1:50 recommended; image 1:100 (datasheet M02792); 9. Primary incubation: Overnight at 4 °C (datasheet M02792). Watch for: Fixation: Keep fixation consistent between sections (standard IHC practice; not target-specific); Caveat: Low IHC–RNA concordance limits tissue predictions (HPA tissue IHC); Isoform / epitope: No listed isoforms; one 1–282 chain (UniProt); Regulation: NPM1 promotes ATF5 degradation (UniProt). Evidence: UniProt Q9Y2D1, HPA tissue IHC · Uncertain, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to…, Datasheet M02792, PMC5067000, PMC8677764. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATF5/immunohistochemistry-ihc-guide.