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AMFR / E3 ubiquitin-protein ligase AMFR · IHC design guide
Design Immunohistochemistry for AMFR
Plan chromogenic IHC-P for AMFR using its generally cytoplasmic tissue staining as a guide (HPA tissue IHC). Start with the catalog antibody at 0.5–1 μg/mL (datasheet PB10039) and compare stained glandular cells with undetected adipocytes (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
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 | General cytoplasmic staining (HPA tissue IHC); ER membrane location (UniProt) | |
| Staining pattern | General cytoplasmic staining across tissue cell types (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 | Keep fixation consistent across samples (standard IHC practice; not target-specific) | |
| Caveat | Adipocytes are undetected despite broad tissue staining (HPA tissue IHC) | |
| Regulation | Widely expressed; no regulator specified (UniProt) | |
| Isoform / epitope | No isoforms; 7 TM spans make epitope side relevant (UniProt) |
Section 1
Recommended AMFR IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet PB10039) with published paraffin-section protocols for renal, prostate, and tumor specimens (PMC4652838; PMC3727633; PMC12625341).
| 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 (datasheet PB10039); test citrate pH 6.0 on matched sections if needed (PMC4652838; PMC3727633).
Section 2
What Is the Expected AMFR Staining Pattern?
AMFR is a seven-pass endoplasmic reticulum (ER) membrane protein (UniProt Q9UKV5 topology and subcellular location). In tissue IHC, expect predominantly cytoplasmic staining in expressing cells, including adrenal and intestinal glandular cells and bronchial respiratory epithelium (HPA tissue IHC). HPA rates the tissue pattern Approved, with medium consistency between antibody staining and RNA expression; interpret intensity alongside controls rather than as a precise measure of abundance (HPA tissue IHC).
| Cytoplasmic color in adrenal glandular cells, colonic or duodenal glandular cells, or bronchial respiratory epithelial cells (HPA: High in each). | This matches HPA's general cytoplasmic pattern and provides an expected positive-cell reference (HPA tissue IHC). Chromogenic IHC may show cytoplasmic color without resolving individual ER membranes (general IHC practice). |
| Predominantly nuclear staining, with little cytoplasmic staining, in an otherwise positive tissue (HPA tissue IHC; UniProt Q9UKV5 subcellular location). | That distribution does not match AMFR's ER membrane location or HPA's cytoplasmic tissue pattern (UniProt Q9UKV5; HPA tissue IHC). Review the primary-antibody and detection controls before assigning it to AMFR (general IHC practice). |
| Strong staining confined to adipocytes, while expected positive cells on the run stain appropriately (HPA: adipocytes Not detected; HPA: High in listed positive cells). | This conflicts with the sampled HPA adipocyte result; cross-reactivity or endogenous detection activity is possible (HPA tissue IHC; general IHC practice). An unexpected cell type alone does not prove artefact because AMFR is widely expressed (UniProt Q9UKV5 tissue specificity). |
| Similar color across cells, extracellular spaces, and tissue-free areas, obscuring cell boundaries (general IHC practice). | Treat this as diffuse background until controls resolve it; it cannot establish the cytoplasmic, cell-associated pattern reported by HPA (HPA tissue IHC; general IHC practice). Assess the no-primary control and detection reagent background (general IHC practice). |
| No detectable color in adrenal, bronchial, colonic, or duodenal positive cells (HPA: High in the named cell populations). | A blank expected-positive control makes a negative test section uninterpretable (general IHC practice). Check section quality and assay performance before inferring absent AMFR; HPA's Approved tissue profile has medium RNA–staining consistency (HPA tissue IHC). |
💡Expected AMFR appearanceCall positive when expected cells show clear cytoplasmic color, potentially strong in HPA High populations; nuclear-only color or color in a no-primary control is suspect (HPA tissue IHC; UniProt Q9UKV5 subcellular location; general IHC practice).
| Compartment and topology (UniProt Q9UKV5) | AMFR occupies the ER membrane and has seven transmembrane segments (UniProt Q9UKV5). HPA describes tissue IHC as generally cytoplasmic, so score the cellular distribution without requiring visible ER outlines in chromogenic sections (HPA tissue IHC; general IHC practice). |
| Tissue contrast (HPA tissue IHC) | HPA reports High staining in specified glandular, respiratory epithelial, glial, and Purkinje cells; adipocytes were Not detected, while oral and vaginal squamous epithelium stained Low (HPA tissue IHC). Choose a documented High population for the positive control, and interpret weak staining in the Low populations accordingly (HPA tissue IHC; general IHC practice). |
| Antibody evidence (HPA antibody validation) | HPA029018, HPA064339, and CAB026381 each have an Approved IHC status (HPA antibody validation). That status supports using their observed pattern as a reference, subject to HPA's stated medium tissue-level consistency; it does not validate an unspecified catalog antibody (HPA tissue IHC; HPA antibody validation). |
| IF/ICC Q&A: where should signal localize? (HPA subcellular ICC-IF) | HPA reports ER localization as Supported and Golgi localization as Approved, with ICC-IF images in A-549 and U2OS (HPA subcellular ICC-IF). IF/ICC can assess those compartments; this tissue IHC interpretation does not establish an IF/ICC protocol (HPA subcellular ICC-IF; general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected-positive cells are blank (HPA: High in selected positive populations). | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. | Repeat with a documented High positive tissue and check the assay's validated IHC-P retrieval, primary-antibody, and detection steps (HPA tissue IHC; general IHC practice). Record whether the positive control recovers before interpreting other negatives (general IHC practice). |
| Unexpected cells show stronger color than documented positive cells (HPA tissue IHC). | Cross-reactivity or endogenous detection activity may contribute (general IHC practice). HPA's adipocyte result is Not detected, but its survey does not prove that every unlisted cell must be negative (HPA tissue IHC). | Compare cellular distribution with a documented positive tissue and a no-primary control; investigate staining that persists without primary antibody as detection background (HPA tissue IHC; general IHC practice). |
| Nuclei dominate the signal (UniProt Q9UKV5 subcellular location; HPA tissue IHC). | Nuclear-dominant staining disagrees with the ER membrane assignment and general cytoplasmic tissue profile (UniProt Q9UKV5; HPA tissue IHC). Its cause cannot be identified from location alone (general IHC practice). | Review staining in expected-positive cells and the no-primary control, then assess antibody specificity if the nuclear pattern persists (HPA tissue IHC; general IHC practice). |
| Brown color covers most of the section or appears outside cells (general IHC practice). | Diffuse reagent background can conceal a cell-associated signal (general IHC practice). It does not match HPA's general cytoplasmic description (HPA tissue IHC). | Inspect a no-primary control, verify washing and detection reagent handling, and reassess whether expected-positive cells remain distinguishable (general IHC practice; HPA tissue IHC). |
| Weak color occurs in oral or vaginal squamous epithelium (HPA: Low in those cell populations). | A weak result can agree with HPA's Low category and does not, by itself, indicate assay failure (HPA tissue IHC). The positive control determines whether the run produced detectable staining (general IHC practice). | Check a documented High tissue on the same run before changing assay conditions; compare like cell types when scoring intensity (HPA tissue IHC; general IHC practice). |
| IF/ICC highlights Golgi as well as ER, while tissue IHC looks broadly cytoplasmic (HPA subcellular ICC-IF; HPA tissue IHC). | HPA assigns Golgi localization Approved and ER localization Supported in ICC-IF, while its tissue IHC summary is general cytoplasmic expression (HPA subcellular ICC-IF; HPA tissue IHC). | Interpret each application against its own HPA reference and controls; do not require the chromogenic section to reproduce the organelle detail of ICC-IF images (HPA subcellular ICC-IF; HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing AMFR staining
PB10039 · IHCIHC analysis of AMFR using anti-AMFR antibody (PB10039). AMFR was detected in a paraffin-embedded section of human placenta tissue. Heat med
PB10039 · IHCIHC analysis of AMFR using anti-AMFR antibody (PB10039). AMFR was detected in paraffin-embedded section of rat kidney tissues. Heat mediated
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.
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: glandular cells must stain (HPA: High in adrenal gland glandular cells). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); on the adrenal slide, morphologically unstained cells should show background only, but they are not validated AMFR-negative cells (UniProt Q9UKV5: widely expressed).
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 control and a concentration-matched rabbit IgG isotype control (caption: rabbit anti-AMFR antibody; standard IHC practice); use AMFR knockout material or a validated peptide block as a biological specificity control (standard IHC practice). For adrenal sections, check endogenous peroxidase and biotin background before interpreting DAB signal (caption: SABC/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected PB10039 tissue-IHC caption does not state a fixative (caption: fixative unreported). Heat retrieval in EDTA at pH 8.0 is documented, but whether retrieval is required is unreported (caption: heat retrieval in EDTA, pH 8.0); available evidence does not establish whether frozen sections or IF are easier, although ICC-IF images exist for A-549 and U2OS (HPA subcellular). Endogenous adrenal pigment can complicate chromogenic scoring, so compare staining with the control section (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 AMFR’s endoplasmic reticulum localisation and the documented paraffin-section method to troubleshoot chromogenic IHC; assess IF separately.
Which retrieval condition should I try first for AMFR in paraffin sections?
Start with heat-mediated EDTA antigen retrieval at pH 8.0 for paraffin sections (datasheet PB10039). This condition preceded AMFR detection in human placenta using 1 μg/ml primary antibody overnight at 4°C (datasheet PB10039). Compare adjacent sections while holding antibody incubation and DAB development constant, so a signal change can be attributed to retrieval (standard IHC practice). If staining is weak, adjust heating duration incrementally and check whether tissue morphology or background deteriorates (standard IHC practice). Judge the result against the expected cytoplasmic, endoplasmic reticulum-associated pattern rather than demanding a crisp cell-surface outline (UniProt Q9UKV5; HPA tissue IHC).
Could fixation explain weak or uneven AMFR staining?
The selected AMFR image documents a paraffin section but does not state its fixative, so target-specific fixation sensitivity is unknown (datasheet PB10039). Record each specimen’s fixative, fixation duration, processing history and section age before comparing staining intensity (standard IHC practice). Test matched sections with the documented EDTA pH 8.0 retrieval and 1 μg/ml primary antibody conditions, changing one processing variable at a time (datasheet PB10039; standard IHC practice). Examine morphology alongside staining: damaged or detached regions cannot support a reliable comparison of antigen preservation (standard IHC practice). Treat any improvement after a fixation change as an observation requiring replication, rather than an established AMFR-specific effect (standard IHC practice).
What AMFR staining pattern is plausible in chromogenic tissue IHC?
Expect predominantly cytoplasmic staining in tissue sections, consistent with the reported general IHC profile (HPA tissue IHC). AMFR is an endoplasmic reticulum membrane protein with 7 transmembrane segments, and palmitoylation promotes its peripheral endoplasmic reticulum localisation (UniProt Q9UKV5). A fine or uneven intracellular pattern can therefore merit review, while an exclusively nuclear pattern needs stronger validation (UniProt Q9UKV5; standard IHC practice). Check that the stain follows intact cells rather than section folds, exposed edges or extracellular deposits (standard IHC practice). Compare suspect fields with adjacent morphology and a control section before assigning compartment-specific expression (standard IHC practice).
How should I assess epitope accessibility in this multipass protein?
AMFR has 7 transmembrane segments and a CUE domain at residues 456–498, making epitope position relevant to accessibility (UniProt Q9UKV5). The supplied record lists 0 isoforms, so it does not support an isoform-specific explanation for divergent tissue staining (UniProt Q9UKV5). Obtain the antibody’s epitope or immunogen location before attributing weak staining to a luminal, cytosolic or membrane-embedded region (standard IHC practice). The record lists phosphoserines at residues 516, 523 and 542, but supplies no evidence that these modifications alter this antibody’s staining (UniProt Q9UKV5). Compare retrieval conditions on adjacent sections before making an epitope-masking claim (standard IHC practice).
How can I cross-check AMFR localisation with multiplex IF?
Use IF as a separate localisation check: AMFR has supported endoplasmic reticulum and approved Golgi localisation in cell imaging (HPA subcellular). Pair AMFR with a marker identifying the expected cell population, then assess signal within those cells rather than relying on tissue-wide overlap (standard IF practice; HPA tissue IHC). Choose fluorophores after inspecting unstained tissue autofluorescence, and use single-stain controls to assess spectral bleed-through (standard IF practice). Determine whether the antibody epitope faces the cytosol or an organelle lumen before selecting permeabilisation conditions for this multipass membrane protein (UniProt Q9UKV5; standard IF practice). Optimise IF fixation independently because the selected paraffin-section caption does not report a fixative or establish IF fixation performance (datasheet PB10039).
What should I check when AMFR DAB staining is diffuse?
First compare the stained section with a no-primary control to identify secondary-reagent or chromogen background (standard IHC practice). The documented workflow used a biotinylated goat anti-rabbit secondary, streptavidin–biotin complex and DAB, so assess reagent-related background under those conditions (datasheet PB10039; standard IHC practice). Apply a peroxidase block as a general chromogenic IHC step, and evaluate whether residual endogenous activity persists in the control (standard IHC practice). The documented section received 10% goat-serum blocking and 1 μg/ml primary antibody overnight at 4°C (datasheet PB10039). If diffuse staining remains, compare carefully washed adjacent sections and check whether signal still follows intact cellular cytoplasm (standard IHC practice; HPA tissue IHC).
How should I score AMFR staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and anatomical region before scoring, because AMFR expression varies among cell types despite low overall tissue specificity (HPA tissue IHC). For chromogenic IHC, report the percentage of positive cells and an intensity-weighted H-score using the same scoring rules across sections (standard IHC practice). If the biological question concerns infiltrating cells, report positive-cell density per mm² of viable tissue instead (standard IHC practice). Normalise comparisons to the eligible cell population or viable tissue area, excluding necrosis, folds and section edges (standard IHC practice). Keep retrieval, antibody incubation, DAB development and image acquisition consistent, and review scores against the documented cytoplasmic staining profile (standard IHC practice; HPA tissue IHC).
How do I distinguish genuine AMFR staining from artefact?
Give greatest weight to intracellular staining in intact cells, consistent with AMFR’s endoplasmic reticulum membrane location and the general cytoplasmic IHC profile (UniProt Q9UKV5; HPA tissue IHC). Compare the cell type with reference patterns: high staining is reported in bronchial respiratory epithelial cells, whereas adipocytes are reported as undetected (HPA tissue IHC). An exclusively nuclear deposit, section-edge accentuation or staining concentrated in necrotic material warrants artefact review (UniProt Q9UKV5; standard IHC practice). Check a no-primary control for residual endogenous enzyme activity or detection-reagent staining before calling a faint result positive (standard IHC practice). Interpret discordant staining cautiously because the tissue IHC evidence is rated approved with medium consistency against RNA expression (HPA tissue IHC).
Boster reagents
Best AMFR / E3 ubiquitin-protein ligase AMFR IHC Antibodies
PB10039 has IHC data from paraffin sections of human placenta, rat kidney and mouse intestine (catalog: IHC captions), plus IF data from T47D cells (catalog: IF caption).
Real IHC data
Anti-AMFR Antibody ®
Cat # PB10039
PB10039 was demonstrated by IHC on paraffin sections of human placenta, rat kidney and mouse intestine (catalog: IHC captions). PB10039 was also demonstrated by IF in T47D cells (catalog: IF caption).
Which to pick: Choose PB10039 for paraffin-section IHC, starting at 0.5–1 μg/ml (datasheet: IHC dilution); its tissue captions do not report a fixative (catalog: IHC captions). For IF/ICC, choose PB10039 at 5 μg/ml (catalog: IF/ICC applications; datasheet: IF dilution), supported by its T47D IF image (catalog: IF caption). PB10039 is also the cross-species choice: the catalog lists human, mouse and rat reactivity, with IHC images from each species (catalog: reactivity and IHC captions); clonality is unreported.
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.
Immunohistochemistry protocol sheet for AMFR (E3 ubiquitin-protein ligase AMFR). Expected localisation General cytoplasmic staining (HPA tissue IHC); ER membrane location (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB10039). Validated IHC antibody: PB10039 at 0.5-1μg/ml (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 placenta tissue; fixative not specified (datasheet PB10039); 2. Fixation: Image fixative and duration unreported (datasheet PB10039); 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 PB10039); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB10039); 8. Primary antibody: Rabbit anti-AMFR, 0.5-1μg/ml (datasheet PB10039); 9. Primary incubation: Overnight at 4 °C (datasheet PB10039). Watch for: Fixation: Keep fixation consistent across samples (standard IHC practice; not target-specific); Caveat: Adipocytes are undetected despite broad tissue staining (HPA tissue IHC); Isoform / epitope: No isoforms; 7 TM spans make epitope side relevant (UniProt); Regulation: Widely expressed; no regulator specified (UniProt). Evidence: UniProt Q9UKV5, HPA tissue IHC · Approved, HPA ICC-IF · Golgi apparatus and endoplasmic reticulum., Datasheet PB10039, PMC4652838, PMC3727633. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/AMFR/immunohistochemistry-ihc-guide.