AATF / Protein AATF · IHC design guide

Design Immunohistochemistry for AATF

Plan AATF paraffin IHC with the catalog antibody at 2 μg/mL (datasheet A03945-3). Score granular cytoplasmic staining (HPA tissue IHC) separately from the nuclear and nucleolar annotation (UniProt), given the uncertain tissue staining reliability (HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for AATF (IHC for AATF): expected localisation Granular cytoplasm in tissue IHC (HPA tissue IHC); nucleus/nucleolus (UniProt), antibody A03945-3, validated IHC image, and IHC protocol steps — Printable **AATF** IHC protocol sheet — expected localisation Granular cytoplasm in tissue IHC (HPA tissue IHC); nucleus/nucleolus (UniProt), antibody A03945-3, controls and protocol steps. Open the full AATF IHC guide →

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AATF 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	Granular cytoplasm in tissue IHC (HPA tissue IHC); nucleus/nucleolus (UniProt)
	Staining pattern	Ubiquitous granular cytoplasmic staining (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A03945-3)
	Positive control ⓘ	Adipose tissue+4 more · see all
	Negative control ⓘ	None in HPA (detected in all 43 tissues); use no-primary + isotype controls

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific)
	Caveat	Tissue pattern conflicts with nuclear annotation (HPA tissue IHC; UniProt)
	Regulation	No expression regulation reported (UniProt)
	Isoform / epitope	No annotated isoforms; chain spans residues 2–560 (UniProt)

Section 1

Recommended AATF IHC & IF Protocols

The catalog antibody protocol uses EDTA retrieval (datasheet: A03945-3); two published AATF IHC protocols provide citrate-based examples (PMC11794051; PMC6168685).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded mouse cerebellum tissue; fixative not specified (datasheet A03945-3)
Fixation	Image fixative and duration unreported (datasheet A03945-3); 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 A03945-3); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A03945-3)
Primary antibody	Rabbit anti-AATF, 1-2 μg/ml (datasheet A03945-3)
Primary incubation	Overnight at 4 °C (datasheet A03945-3)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A03945-3)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	AATF-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: Ubiquitous granular cytoplasmic expression. No signal in the no-primary control.

💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet: A03945-3); citrate retrieval was reported with different antibodies (PMC11794051; PMC6168685).

Section 2

What Is the Expected AATF Staining Pattern?

AATF is nuclear and nucleolar (UniProt Q9NY61), and HPA ICC-IF mainly places it in nucleoli (HPA subcellular). Tissue IHC instead reports ubiquitous granular cytoplasmic staining, with Uncertain reliability because that location conflicts with external data (HPA tissue IHC). Signal may occur across many cell types (UniProt Q9NY61 tissue specificity); the cytoplasmic IHC pattern needs independent confirmation before it is assigned to AATF.

What am I looking at on my slide?
Discrete nuclear staining, strongest in nucleoli, within identifiable cells.	This fits the annotated location (UniProt Q9NY61; HPA subcellular ICC-IF). For paraffin IHC, confirm specificity with independent evidence: HPA rates its tissue IHC location Uncertain (HPA tissue IHC).
Granular cytoplasmic staining dominates while nuclei remain unstained.	HPA reports this tissue IHC pattern but flags its location as conflicting with external data (HPA tissue IHC). Treat it as unresolved staining, not proof of cytoplasmic AATF (UniProt Q9NY61; HPA subcellular).
Strong staining appears in an unexpected cell type or follows tissue boundaries indiscriminately.	Check for cross-reactivity or endogenous detection activity (general IHC practice). AATF is broadly expressed, so cell type alone cannot establish an error (UniProt Q9NY61); compare compartment, morphology and controls.
Diffuse color covers cells and surrounding tissue without clear intracellular boundaries.	This is difficult to score as AATF because its annotated locations are intracellular (UniProt Q9NY61; HPA subcellular). Review background controls, blocking, detection and chromogen development (general IHC practice).
No signal appears in a section selected from an HPA high-staining tissue.	Check assay performance before calling AATF absent: HPA lists high staining in several tissues, but its IHC location assessment is Uncertain (HPA tissue IHC). Confirm tissue preservation and detection with suitable controls (general IHC practice).

💡Expected AATF appearanceA credible positive shows localized nuclear, particularly nucleolar, staining in identifiable cells (UniProt Q9NY61; HPA subcellular); intensity needs assay-specific controls, while dominant granular cytoplasm remains unresolved because HPA tissue IHC is Uncertain (HPA tissue IHC).

How each factor affects the staining
Compartment evidence	UniProt places AATF in nucleus and nucleolus; HPA ICC-IF supports nucleoli and also records nucleoplasm (UniProt Q9NY61; HPA subcellular). HPA tissue IHC reports a conflicting cytoplasmic pattern (HPA tissue IHC).
Tissue selection	High HPA staining is reported in adipocytes and several epithelial, neuronal and endothelial populations; parathyroid glandular cells are listed as low (HPA tissue IHC). These are comparative staining observations, not specificity controls.
Antibody evidence	One listed antibody has Uncertain IHC status (HPA004940), while another has Supported ICC status (HPA075963; HPA antibodies). Evidence for one assay or antibody does not validate the other.
Protein architecture	AATF has no transmembrane segment or annotated signal peptide, propeptide or isoforms (UniProt Q9NY61). These annotations support an intracellular interpretation but do not establish IHC epitope access or retrieval conditions.
IF/ICC question: should its pattern settle the IHC call?	No. HPA ICC-IF supports mainly nucleolar staining, with additional nucleoplasm (HPA subcellular), while tissue IHC is Uncertain (HPA tissue IHC). Assess the paraffin-section result on its own controls.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Nuclear or nucleolar signal is weak or absent.	The assay may lack detectable signal; target-specific fixation sensitivity is unreported in the supplied sources.	Check a tissue with HPA high staining, the positive detection control and the counterstain; then optimize retrieval and antibody conditions as general IHC practice (HPA tissue IHC).
Only granular cytoplasmic staining is visible.	This matches HPA's reported tissue pattern, whose location is Uncertain and conflicts with other localization evidence (HPA tissue IHC; UniProt Q9NY61).	Record it as unresolved; compare with an independent antibody or orthogonal evidence before assigning the compartment (general IHC practice).
Signal appears in nearly every compartment or cell outline.	Excess background, cross-reactivity or endogenous detection activity may obscure intracellular localization (general IHC practice).	Inspect negative and detection-only controls; adjust blocking, antibody concentration and chromogen development using standard IHC controls (general IHC practice).
A purported negative tissue shows staining.	Broad AATF expression makes an assumed negative tissue unreliable (UniProt Q9NY61); HPA lists low parathyroid staining, not an absence of protein (HPA tissue IHC).	Use reagent controls to assess background, and interpret tissue comparisons alongside compartment and cell morphology (general IHC practice).
An HPA high-staining tissue gives no signal.	The tissue comparison may fail because of assay performance; HPA high staining is observational and its IHC reliability is Uncertain (HPA tissue IHC).	Verify section integrity and detection controls, then optimize retrieval and antibody conditions without assuming a target-specific fixation effect (general IHC practice).
IF/ICC shows nucleoli but IHC shows cytoplasm.	HPA assigns supported ICC evidence and uncertain tissue IHC evidence to different listed antibodies (HPA antibodies; HPA tissue IHC).	Report the assay and antibody separately; seek independent validation before treating the IHC cytoplasmic pattern as AATF localization (general IHC practice).

Source: UniProt Q9NY61 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing AATF staining

IHC analysis of AATF using anti-AATF antibody (A03945-3). AATF was detected in a paraffin-embedded section of mouse cerebellum tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-AATF Antibody (A03945-3) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A03945-3 · IHCIHC analysis of AATF using anti-AATF antibody (A03945-3). AATF was detected in a paraffin-embedded section of mouse cerebellum tissue. Heat

IF analysis of AATF using anti-AATF antibody (A03945-3) and anti-Beta Tubulin antibody (M01857-3). AATF was detected in immunocytochemical section of A549 cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-AATF Antibody (A03945-3) and mouse anti-Beta Tubulin antibody (M01857-3) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) and Cy3 Conjugated Goat Anti-Mouse IgG (BA1031) were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A03945-3 · IFIF analysis of AATF using anti-AATF antibody (A03945-3) and anti-Beta Tubulin antibody (M01857-3). AATF was detected in immunocytochemical s

A03945 · IHCImmunohistochemistry of AATF in human small intestine tissue with AATF antibody at 10 μg/mL.

A03945 · IFImmunofluorescence of AATF in Human Small Intestine cells with AATF antibody at 20 μg/mL.

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000275700-AATF/tissue/cerebral+cortex#imid_1995816

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000275700-AATF/tissue/colon#imid_1995750

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000275700-AATF/tissue/liver#imid_1995751

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000275700-AATF/tissue/kidney#imid_1995744

HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA075963; green = AATF, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for AATF IHC & IF

🧪Run adipose tissue first and score staining in adipocyte nuclei, the HPA-listed cell type with High AATF expression (HPA: High in adipocytes; UniProt Q9NY61: nucleus, nucleolus). HPA detects AATF in all 43 scored tissues, so there is no validated negative tissue or internal negative cell population; no-primary and isotype controls provide the negative comparison, with no specific cellular staining expected in either (HPA: no negative tissue rows; standard IHC practice).

Positive control tissue: Adipose tissue (Adipocytes, HPA High)

Negative control tissue: None in HPA: AATF is detected in all 43 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AATF in A-549, U-251MG, U2OS, with annotated localisation: Nucleoli (supported) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only slide and a rabbit IgG control matched to the primary antibody’s class and clonality where applicable; knockout tissue, if available, can provide a biological specificity control (hero caption: rabbit primary; standard IHC practice). Quench endogenous peroxidase and check for background around adipose tissue and blood before interpreting DAB staining (standard chromogenic IHC practice).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A03945-3 paraffin-section caption does not state a fixative (hero caption: fixative unreported). That caption uses heat retrieval in EDTA at pH 8.0, which is a starting condition but does not establish that retrieval is required; the supplied evidence does not establish that frozen sections or IF/ICC are easier than paraffin IHC (hero caption: EDTA retrieval; HPA: ICC-IF images). In adipose tissue, score intact nuclei rather than empty lipid vacuoles, which can appear unstained after tissue processing (standard histology practice).

HPA tissue IHC evidence for AATF

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Subcellular location is contradicted by external 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
Adipose tissue	Adipocytes	High	Protein (IHC)	HPA →
Adrenal gland	Glandular cells	High	Protein (IHC)	HPA →
Appendix	Glandular cells	High	Protein (IHC)	HPA →
Breast	Glandular cells	High	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
None in HPA: AATF is detected in all 43 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced AATF IHC Tips

Troubleshoot AATF staining in paraffin sections by checking retrieval, nuclear localisation, and controls before interpreting chromogenic signal.

What retrieval conditions should I start with for AATF in paraffin sections?

Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A03945-3). The catalog antibody stained a paraffin-embedded mouse cerebellum section after this retrieval, followed by 2 μg/ml primary antibody overnight at 4°C (caption A03945-3). If staining is weak, first compare sections processed with consistent heating, cooling, and wash conditions, then titrate retrieval time while monitoring tissue integrity (standard IHC practice). Keep a known positive section in each run and judge improvement by nuclear or nucleolar signal, because AATF localises to the nucleus and nucleolus (UniProt Q9NY61; HPA subcellular).

Could fixation explain weak or uneven AATF staining?

Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state its fixative (caption A03945-3). Record the fixative, fixation duration, section age, and processing conditions for each specimen, then compare matched sections under the same retrieval and antibody conditions (standard IHC practice). If staining differs across batches, use a consistently processed positive control to distinguish a processing change from a staining-run change (standard IHC practice). Assess preserved nuclear detail alongside DAB signal, since AATF is reported in the nucleus and nucleolus; tissue patterns alone cannot establish a fixation effect (UniProt Q9NY61; HPA subcellular; standard IHC practice).

How should I assess cytoplasmic AATF staining when nucleolar staining is expected?

Score nuclear and nucleolar signal separately from cytoplasmic signal: AATF is assigned to the nucleus and nucleolus, with supported nucleolar localisation in cell images (UniProt Q9NY61; HPA subcellular). HPA tissue IHC instead describes ubiquitous granular cytoplasmic expression and rates its tissue staining Uncertain because subcellular location conflicts with external data (HPA tissue IHC). Check whether cytoplasmic DAB persists in a no-primary control and whether nuclear detail is preserved before calling it specific (standard IHC practice). Report discordant compartment patterns explicitly, including the fraction of stained cells and the control results, rather than combining them into one positive score (standard IHC practice).

Can isoforms or epitope processing account for different AATF staining patterns?

The supplied record lists 0 isoforms and a protein chain spanning residues 2–560, so an isoform-specific staining explanation is unsupported here (UniProt Q9NY61). It also lists modified residues, including phosphoserines at 61, 63, 150, and 155, but supplies no antibody epitope or evidence that these modifications alter staining (UniProt Q9NY61; caption A03945-3). Compare serial sections using the same retrieval and detection conditions before attributing a pattern shift to epitope accessibility (standard IHC practice). If the epitope remains undisclosed, describe compartment and cell-level results without assigning a molecular cause to discordant staining (standard IHC practice).

How can IF help assess an uncertain AATF IHC pattern?

Use IF as a separate localisation check, because cell-image evidence places AATF mainly in nucleoli and additionally in nucleoplasm (HPA subcellular). For multiplexing, pair AATF with a validated marker for the cell type being assessed and use distinct fluorophores whose channels can be separated (standard IF practice). Select a fluorophore channel after checking the specimen’s autofluorescence, and include single-stain and no-primary controls to identify bleed-through and background (standard IF practice). AATF has no annotated transmembrane segment; for an intracellular nuclear epitope, optimise permeabilisation alongside fixation so the antibody can enter cells while nucleolar structure remains interpretable (UniProt Q9NY61; standard IF practice).

What should I check when AATF DAB staining is diffuse or granular?

Run a no-primary control and inspect endogenous pigment, tissue folds, and section edges before adjusting antibody concentration (standard IHC practice). The selected caption used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, a peroxidase-conjugated secondary for 30 minutes at 37°C, and DAB development (caption A03945-3). Apply an endogenous peroxidase block and compare DAB development times across matched sections to assess enzyme-related background (standard chromogenic IHC practice). Treat granular cytoplasmic staining cautiously: HPA reports that tissue pattern but rates it Uncertain, while independent localisation evidence supports nucleoli and nucleoplasm (HPA tissue IHC; HPA subcellular).

How should I quantify AATF staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring, then record nuclear or nucleolar positivity separately from cytoplasmic DAB (UniProt Q9NY61; HPA subcellular; standard IHC practice). For a defined population, report the percentage of positive cells and an H-score based on intensity and the percentage at each intensity; keep thresholds fixed across slides (standard IHC practice). If cells are counted by area, report positive-cell density per mm² and normalise to evaluable tissue area, excluding folds and necrosis consistently (standard IHC practice). Include a matched positive control in each run, and do not pool HPA’s uncertain cytoplasmic pattern with compartment-specific nuclear scores (HPA tissue IHC; standard IHC practice).

When is an AATF-positive IHC result convincing rather than artefactual?

Give greatest weight to reproducible nuclear or nucleolar staining in intact cells, since those are the reported AATF compartments (UniProt Q9NY61; HPA subcellular). Compare the candidate signal with a no-primary control and a consistently processed positive section, and check that counterstaining permits identification of the stained cell type (standard IHC practice). Discount staining restricted to section edges, folds, or necrotic regions, and investigate diffuse DAB that could reflect endogenous peroxidase activity (standard IHC practice). High staining is reported in cerebellar molecular-layer cells, but HPA rates its tissue IHC localisation Uncertain; identify the cells and report cytoplasmic discordance rather than treating either pattern as definitive (HPA tissue IHC).

Boster reagents

Best AATF / Protein AATF IHC Antibodies

Two anti-AATF antibodies have IHC images from human small intestine and mouse cerebellum, plus IF images from human small intestine cells, A549 cells, and rat brain (catalog image captions).

Real IHC data

Anti-AATF Antibody ®

Cat # A03945-3

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Real IHC data Immunohistochemistry of AATF in human small intestine tissue with AATF antibody at 10 μg/mL.

Anti-Protein AATF AATF Antibody

Cat # A03945

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A03945 has IHC-P data from human small intestine and IF data from human small intestine cells (catalog: applications and image captions). A03945-3 has IHC data from paraffin-embedded mouse cerebellum and IF data from A549 cells and paraffin-embedded rat brain (catalog: applications and image captions).

Which to pick: For paraffin-section IHC, choose A03945-3 when its documented mouse cerebellum workflow is relevant: EDTA retrieval at pH 8.0 and 2 μg/mL primary antibody; choose A03945 for the documented human small intestine IHC example at 10 μg/mL (each SKU’s IHC image caption). For IF/ICC or work spanning human, mouse, and rat, choose A03945-3 based on its listed applications, species reactivity, and IF images; clone type is unreported for both SKUs (catalog: applications, reactivity, IF image captions, clone fields). Fixative is unreported in both IHC captions (catalog: IHC image captions).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry Q9NY61 (AATF_HUMAN, Protein AATF).
Human Protein Atlas. AATF tissue IHC expression (reliability: Uncertain).
Human Protein Atlas. AATF subcellular location (ICC-IF): Mainly localized to the nucleoli. In addition localized to the nucleoplasm..
Human Protein Atlas. AATF antibody validation summary (2 antibodies).
Apoptosis antagonizing transcription factor expression and its validation as a potential diagnostic and prognostic biomarker in oral squamous cell carcinoma. Frontiers in oncology 2025 — PMC11794051.
The PI3K/AKT axis modulates AATF activity in Wilms' tumor cells. FEBS open bio 2018 — PMC6168685.
The Oncogenic and Immunological Roles of Apoptosis Antagonistic Transcription Factors in Human Tumors: A Pan-Cancer Analysis. Oxidative medicine and cellular longevity 2022 — PMC9581705.
AATF and SMARCA2 are associated with thyroid volume in Hashimoto's thyroiditis patients. Scientific reports 2020 — PMC7000742.
PubMed PMID:11027528 — UniProt-cited evidence.
PubMed PMID:10783144 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.