AMFR / E3 ubiquitin-protein ligase AMFR · IHC design guide

Design Immunohistochemistry for AMFR

Plan AMFR IHC in paraffin sections using the observed general cytoplasmic pattern (HPA tissue IHC). Compare high staining in colon glandular cells with undetected adipocytes in adipose tissue (HPA tissue IHC), and start with 0.5–1 μg/mL catalog antibody (datasheet: PB10039).

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

Immunohistochemistry protocol sheet for AMFR (IHC for AMFR): expected localisation Cytoplasmic tissue staining (HPA tissue IHC); ER membrane protein (UniProt), antibody PB10039, validated IHC image, and IHC protocol steps — Printable **AMFR** IHC protocol sheet — expected localisation Cytoplasmic tissue staining (HPA tissue IHC); ER membrane protein (UniProt), antibody PB10039, controls and protocol steps. Open the full AMFR IHC guide →

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AMFR 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 tissue staining (HPA tissue IHC); ER membrane protein (UniProt)
	Staining pattern	General cytoplasmic staining across tissues (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet PB10039)
	Positive control ⓘ	Adrenal gland+4 more · see all
	Negative control ⓘ	Adipose tissue

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 PB10039)
	Caveat	Antibody staining has medium consistency with RNA data (HPA tissue IHC)
	Regulation	No specific expression regulator reported (UniProt)
	Isoform / epitope	0 isoforms; check epitope side across 7 TM segments (UniProt)

Section 1

Recommended AMFR IHC & IF Protocols

The catalog antibody’s IHC-P protocol (datasheet PB10039) is accompanied by four published AMFR IHC protocols (PMC4652838; PMC3727633; PMC12625341; PMC2575378).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human placenta tissue; fixative not specified (datasheet PB10039)
Fixation	Image fixative and duration unreported (datasheet PB10039); 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 PB10039); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet PB10039)
Primary antibody	Rabbit anti-AMFR, 0.5-1μg/ml (datasheet PB10039)
Primary incubation	Overnight at 4 °C (datasheet PB10039)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB10039)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	AMFR-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet PB10039). The published paraffin protocols used citrate retrieval (PMC4652838; PMC3727633; PMC12625341).

Section 2

What Is the Expected AMFR Staining Pattern?

AMFR is a seven-pass endoplasmic reticulum membrane protein (UniProt Q9UKV5 topology). In paraffin-section IHC, expect predominantly cytoplasmic staining, including strong staining in colon glandular cells and bronchial respiratory epithelial cells (HPA tissue IHC: High). HPA rates the tissue pattern Approved, with medium consistency between antibody staining and RNA expression (HPA tissue IHC: reliability).

What am I looking at on my slide?
Strong cytoplasmic staining in colon glandular cells or bronchial respiratory epithelial cells, with nuclei comparatively clear.	This fits the reported cell types and intensity (HPA tissue IHC: High in both). A cytoplasmic pattern is consistent with AMFR at the endoplasmic reticulum membrane (UniProt Q9UKV5 subcellular location); chromogenic IHC alone does not resolve individual organelles (general IHC practice).
Predominantly nuclear staining, or a crisp surface-only outline without a convincing cytoplasmic component.	That distribution conflicts with the reported general cytoplasmic IHC pattern (HPA tissue IHC) and endoplasmic reticulum membrane location (UniProt Q9UKV5). Treat it as suspect and compare with controls; compartment mismatch alone does not identify its cause (general IHC practice).
Strong staining confined to adipocytes, while the expected cells in a positive reference section do not stain.	HPA reports AMFR as not detected in adipocytes and High in colon glandular cells (HPA tissue IHC). Consider off-target staining or endogenous detection activity (general IHC practice). Interpret adipocytes specifically: the HPA result does not designate every cell in adipose tissue as negative.
Diffuse color covers the section and obscures cell borders, including areas without the expected cell-specific pattern.	A section-wide haze is difficult to reconcile with interpretable cytoplasmic staining (HPA tissue IHC: general cytoplasmic expression). Background from detection reagents or insufficient blocking is possible (general IHC practice); the appearance by itself does not establish AMFR expression.
No staining appears in colon glandular cells on a reference section.	Those cells are reported High (HPA tissue IHC), so check the run before interpreting a study sample as negative (general IHC practice). HPA reports medium consistency between staining and RNA data (HPA tissue IHC: reliability); one reference specimen cannot guarantee a positive result in every preparation.

💡Expected AMFR appearanceCall a positive result when expected cells show predominantly cytoplasmic staining, potentially strong in colon glandular cells (HPA tissue IHC: High); isolated nuclear staining or uniform haze is suspect (HPA tissue IHC: general cytoplasmic pattern; general IHC practice).

How each factor affects the staining
Cell type used as a reference	Colon glandular and bronchial respiratory epithelial cells are High, oral mucosal squamous cells are Low, and adipocytes are Not detected (HPA tissue IHC). Compare the named cell populations rather than assigning one expected intensity to an entire section.
Membrane topology and compartment	AMFR has seven transmembrane segments and localizes to the endoplasmic reticulum membrane (UniProt Q9UKV5). This supports a cytoplasmic IHC interpretation, but neither topology nor staining level establishes AMFR-specific sensitivity to fixation or antigen retrieval.
Strength of pattern evidence	HPA calls the tissue IHC pattern Approved and reports medium consistency with RNA expression (HPA tissue IHC: reliability). Its listed IHC antibodies are Approved, not designated Enhanced in the supplied antibody record (HPA antibodies). Treat a surprising result as a reason to check controls, not as proof of absence.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A reference section expected to stain is blank.	Colon glandular cells are reported High (HPA tissue IHC); a blank run may reflect a staining or detection problem (general IHC practice).	Confirm the reference cells are present, then review antibody application, antigen retrieval, and detection with a working positive control (general IHC practice). No AMFR-specific retrieval condition is supplied.
The slide shows broad, low-contrast brown haze.	Diffuse haze does not match the reported general cytoplasmic pattern (HPA tissue IHC); nonspecific reagent binding or endogenous activity may contribute (general IHC practice).	Review blocking and detection controls, and compare the haze with staining in the expected cells (general IHC practice; HPA tissue IHC: High in colon glandular cells).
Adipocytes appear strongly positive.	HPA reports AMFR as Not detected in adipocytes (HPA tissue IHC). Off-target binding or endogenous detection activity is possible (general IHC practice).	Check a reagent control and a known positive cell population on the same run before assigning the adipocyte signal to AMFR (general IHC practice; HPA tissue IHC: High in colon glandular cells).
Staining is mainly nuclear or outlines only the cell surface.	The pattern differs from general cytoplasmic tissue staining (HPA tissue IHC) and endoplasmic reticulum membrane localization (UniProt Q9UKV5).	Recheck compartment assignment against the counterstain and inspect controls for nonspecific signal (general IHC practice). Do not score the unexpected compartment as a confirmed AMFR pattern.
A low-staining sample is being called AMFR negative.	HPA reports Low staining in oral and vaginal squamous epithelial cells, alongside low tissue specificity overall (HPA tissue IHC). A faint result needs a working reference to interpret.	Confirm staining in a High reference cell population and compare cell-specific signal with background before scoring the sample (HPA tissue IHC: High in colon glandular cells; general IHC practice).
Can IF/ICC help assess an uncertain IHC compartment?	HPA reports endoplasmic reticulum localization as Supported and Golgi localization as Approved in ICC-IF (HPA subcellular).	Use that localization as context for the IHC interpretation; consult the separate IF/ICC guide for its experiment design (HPA subcellular; UniProt Q9UKV5: endoplasmic reticulum membrane).

Source: UniProt Q9UKV5 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing AMFR staining

IHC analysis of AMFR using anti-AMFR antibody (PB10039). AMFR was detected in a paraffin-embedded section of human placenta 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 1 μg/ml rabbit anti-AMFR Antibody (PB10039) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

PB10039 · IHCIHC analysis of AMFR using anti-AMFR antibody (PB10039). AMFR was detected in a paraffin-embedded section of human placenta tissue. Heat med

IHC analysis of AMFR using anti-AMFR antibody (PB10039). AMFR was detected in paraffin-embedded section of rat kidney tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-AMFR Antibody (PB10039) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

PB10039 · IHCIHC analysis of AMFR using anti-AMFR antibody (PB10039). AMFR was detected in paraffin-embedded section of rat kidney tissues. Heat mediated

IF analysis of AMFR using anti-AMFR antibody (PB10039). AMFR was detected in an immunocytochemical section of T47D cells. 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-AMFR Antibody (PB10039) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 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.

PB10039 · IFIF analysis of AMFR using anti-AMFR antibody (PB10039). AMFR was detected in an immunocytochemical section of T47D cells. Enzyme antigen ret

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000159461-AMFR/tissue/cerebral+cortex#imid_21231035

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000159461-AMFR/tissue/colon#imid_21230945

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000159461-AMFR/tissue/liver#imid_21230962

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000159461-AMFR/tissue/kidney#imid_21230955

HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA077835; green = AMFR, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for AMFR IHC & IF

🧪Run adrenal gland first: its glandular cells should stain for AMFR (HPA: High in adrenal gland glandular cells). Use adipose tissue as the negative tissue, where adipocytes are listed as not detected (HPA: Not detected in adipocytes); on the adrenal slide, cells without specific staining should show counterstain alone, but should not be assumed to be biological negatives without validation (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 AMFR in A-549, U2OS, with annotated localisation: Endoplasmic reticulum (supported), Golgi apparatus (approved) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only slide, a matched nonimmune rabbit IgG isotype control, and AMFR-knockout material as a biological negative (selected-SKU caption: rabbit primary; standard IHC practice). For the caption’s biotin-based DAB detection, quench endogenous peroxidase and assess endogenous biotin staining in adrenal tissue (selected-SKU caption: SABC and DAB; standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative not stated). The reported IHC procedure uses heat retrieval in EDTA at pH 8.0; whether retrieval is strictly required, or frozen sections or IF are easier, is unreported (selected-SKU caption: EDTA retrieval; HPA: ICC-IF images in A-549 and U2OS). Endogenous peroxidase or biotin staining could complicate interpretation of the biotin-based DAB readout in adrenal tissue, so assess those signals with the technical controls (selected-SKU caption: SABC and DAB; standard IHC practice).

HPA tissue IHC evidence for AMFR

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
Adrenal gland	Glandular cells	High	Protein (IHC)	HPA →
Appendix	Glandular cells	High	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	High	Protein (IHC)	HPA →
Caudate	Glial cells	High	Protein (IHC)	HPA →
Cerebellum	Purkinje 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 AMFR IHC Tips

Use the documented paraffin-section workflow as a starting point, then assess AMFR staining by cell type, subcellular pattern, and matched controls.

Which retrieval condition should I start with for AMFR paraffin sections?

Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for the catalog antibody (datasheet PB10039). The documented human placenta section was subsequently stained with 1 μg/ml primary antibody overnight at 4°C, so reproduce those conditions when comparing retrieval results (datasheet PB10039). If staining is weak, compare a shorter and longer heating interval on adjacent sections while keeping antibody concentration, detection, and imaging settings fixed (standard IHC practice). Evaluate preservation alongside signal: damaged morphology or staining concentrated at section edges makes an apparent gain in intensity unreliable (standard IHC practice).

How should I troubleshoot fixation-related loss of AMFR staining?

The selected paraffin-section caption does not report a fixative, so AMFR-specific sensitivity to fixation is unknown (datasheet PB10039). Record the specimen’s actual fixative and duration, and compare similarly processed sections before attributing a weak result to fixation (standard IHC practice). Run the documented EDTA pH 8.0 retrieval and 1 μg/ml primary condition on a section with intact morphology as a reference (datasheet PB10039). If processing histories differ, assess morphology and staining together; neither tissue expression patterns nor AMFR’s 7 transmembrane segments establish a target-specific fixation effect (HPA tissue IHC; UniProt Q9UKV5 topology).

What staining pattern is plausible for AMFR in chromogenic IHC?

Expect predominantly cytoplasmic staining in tissue sections, while remembering that AMFR is an endoplasmic reticulum membrane protein (HPA tissue IHC; UniProt Q9UKV5 subcellular location). Its reported cellular locations include supported endoplasmic reticulum and approved Golgi apparatus staining, so a perinuclear cytoplasmic accent can be plausible when morphology supports it (HPA subcellular). Compare glandular cells or respiratory epithelial cells with adjacent tissue compartments on the same slide, rather than calling diffuse chromogen throughout every structure positive (HPA tissue IHC; standard IHC practice). Crisp nuclear-only staining warrants a detection and specificity check because it does not fit the reported AMFR locations (UniProt Q9UKV5 subcellular location; HPA subcellular).

Could epitope location explain inconsistent AMFR staining across sections?

AMFR has 7 annotated transmembrane segments, a CUE domain at residues 456–498, and no listed isoforms in this record (UniProt Q9UKV5 topology, domains, isoforms). Those features make antibody epitope placement relevant, but the supplied catalog caption does not identify the recognized residues (datasheet PB10039). Check the antibody’s documented immunogen or epitope before assigning a loss of staining to domain masking or post-translational modification (standard IHC practice). Phosphoserines at 516, 523, and 542 are annotated, yet these annotations alone do not show that phosphorylation affects this antibody’s paraffin-section staining (UniProt Q9UKV5 modified residues; datasheet PB10039).

How can IF help assess a questionable AMFR IHC pattern?

Use IF as a separate localisation check: pair AMFR with a marker for the expected cell population, such as respiratory epithelium, and ask whether signals occur in the same cells (HPA tissue IHC; standard IF practice). Include a longer-wavelength fluorophore and inspect an unstained specimen channel when tissue autofluorescence obscures the signal (standard IF practice). AMFR spans the membrane 7 times; select permeabilisation only after establishing which side contains this antibody’s epitope, since the supplied caption does not map it (UniProt Q9UKV5 topology; datasheet PB10039). Compare the IF compartment with the endoplasmic reticulum and Golgi locations before using it to interpret chromogenic staining (HPA subcellular).

How do I separate AMFR signal from chromogenic background?

First examine a section processed without primary antibody; persistent brown signal points toward detection chemistry or endogenous tissue activity (standard IHC practice). The documented method uses a biotinylated secondary, streptavidin–biotin complex, and DAB, making matched detection-only controls particularly useful (datasheet PB10039; standard IHC practice). Apply the routine peroxidase block and appropriate nonspecific-binding block, then compare background with the documented 10% goat-serum block and 1 μg/ml primary condition (standard IHC practice; datasheet PB10039). Treat diffuse staining over empty spaces, folded sections, or tissue edges cautiously, even if expected cell populations also stain (standard IHC practice; HPA tissue IHC).

How should I score AMFR IHC across heterogeneous tissue? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and a consistent positivity threshold before scoring, then record the percentage of positive cells and an H-score using intensity grades 0–3 (standard IHC practice). Score cytoplasmic staining in comparable viable regions because the tissue profile describes general cytoplasmic expression (HPA tissue IHC). Normalise positive-cell counts to all evaluable cells of the chosen type, or report positive-cell density per mm² of evaluable tissue when area is the denominator (standard IHC practice). Keep retrieval, exposure to DAB, and scoring rules consistent across sections, and exclude folds, necrosis, and tissue edges from the measurement region (standard IHC practice).

When should I distrust an apparent AMFR-positive region?

A credible positive region shows cytoplasmic staining in identifiable cells, consistent with the tissue profile and AMFR’s endoplasmic reticulum membrane location (HPA tissue IHC; UniProt Q9UKV5 subcellular location). High staining has been reported in bronchial respiratory epithelial cells and several glandular cell populations, whereas adipocytes were reported as not detected; use these as context, not absolute controls for every specimen (HPA tissue IHC). Question nuclear-only signal, staining restricted to section edges or necrosis, and pigment that persists without primary antibody (UniProt Q9UKV5 subcellular location; standard IHC practice). Because the tissue-IHC reliability is approved with medium RNA–staining consistency, resolve discordant findings with morphology and matched controls (HPA tissue IHC; standard IHC practice).

Boster reagents

Best AMFR / E3 ubiquitin-protein ligase AMFR IHC Antibodies

PB10039 has IHC images from paraffin sections of human placenta, rat kidney and mouse intestine, plus an ICC/IF image from T47D cells (catalog image captions).

Real IHC data

Anti-AMFR Antibody ®

Cat # PB10039

View product →

PB10039 is listed for IHC, IF and ICC in human, mouse and rat samples (catalog applications and reactivity). Its IHC images show human placenta, rat kidney and mouse intestine; its ICC/IF image shows T47D cells (catalog image captions).

Which to pick: For tissue IHC, choose PB10039: its own images document paraffin sections stained at 1 μg/ml; the fixative is unreported (PB10039 IHC image captions). For IF/ICC, choose PB10039 at the listed 5 μg/ml, supported by its T47D cell image (PB10039 IF image caption; catalog dilution). For work across species, PB10039 lists human, mouse and rat reactivity; clonality is unreported (catalog reactivity; catalog clone field).

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

References

UniProt Consortium. UniProt entry Q9UKV5 (AMFR_HUMAN, E3 ubiquitin-protein ligase AMFR).
Human Protein Atlas. AMFR tissue IHC expression (reliability: Approved).
Human Protein Atlas. AMFR subcellular location (ICC-IF): Localized to the Golgi apparatus and endoplasmic reticulum..
Human Protein Atlas. AMFR antibody validation summary (4 antibodies).
Increased Expression of the Autocrine Motility Factor is Associated With Poor Prognosis in Patients With Clear Cell-Renal Cell Carcinoma. Medicine 2015 — PMC4652838.
Enhanced expression of SOS1 is detected in prostate cancer epithelial cells from African-American men. International journal of oncology 2009 — PMC3727633.
AMFR-mediated ER-phagy regulation and therapeutic targeting in osteosarcoma: a multifunctional nanoplatform strategy. Journal of nanobiotechnology 2025 — PMC12625341.
Raft-dependent endocytosis of autocrine motility factor/phosphoglucose isomerase: a potential drug delivery route for tumor cells. PloS one 2008 — PMC2575378.
PubMed PMID:10456327 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.
PubMed PMID:7626106 — UniProt-cited evidence.