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MANF / Mesencephalic astrocyte-derived neurotrophic factor · IHC design guide
Design Immunohistochemistry for MANF
Plan MANF chromogenic IHC in paraffin sections using high-staining colon glandular cells and not-detected skeletal-muscle myocytes as references (HPA tissue IHC). The IHC-validated antibody A03825 lists a dilution of 1:100–1:300 (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MANF 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 lumen (UniProt) | |
| Staining pattern | Cytoplasmic staining in several tissues, highest in immune cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Bone marrow+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
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 A03825) | |
| Caveat | Secretion may separate protein staining from RNA expression (HPA tissue IHC) | |
| Regulation | ER stress or hypoxia up-regulates MANF (UniProt) | |
| Isoform / epitope | No annotated isoforms; mature chain 25–182 has no membrane span (UniProt) |
Section 1
Recommended MANF IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by four published MANF IHC methods (PMC10750294; PMC6851024; PMC9953319; PMC3938758). The rat brain method uses frozen sections (PMC3938758: methods).
| Sample | Paraffin-embedded rat brain tissue; fixative not specified (datasheet A03825) |
| Fixation | Image fixative and duration unreported (datasheet A03825); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-MANF, 1:100-1:300 (datasheet A03825) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | MANF-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues, highest in immune cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen retrieval); the pancreatic study used high-pH retrieval (PMC9953319: methods).
Section 2
What Is the Expected MANF Staining Pattern?
MANF is retained in the ER and sarcoplasmic reticulum under normal conditions and can be secreted during ER stress or hypoxia; it has no transmembrane segment (UniProt P55145). In tissue IHC, expect cytoplasmic staining, with high staining reported in selected hematopoietic, neuronal, Purkinje and glandular cells (HPA tissue IHC). HPA rates the tissue pattern Approved, with medium consistency between staining and RNA data (HPA tissue IHC).
| Clear cytoplasmic staining in bone marrow hematopoietic cells or cerebellar Purkinje cells. | This fits two reported high cell-specific IHC patterns (HPA tissue IHC). ER retention supports an intracellular pattern (UniProt P55145); assess the named cells rather than averaging the whole tissue. |
| Signal is predominantly nuclear, with little cytoplasmic staining. | A nuclear-dominant pattern does not match the reported cytoplasmic IHC pattern or supported ER location (HPA tissue IHC; HPA ICC-IF). Check specificity and counterstain interpretation before scoring it as MANF. |
| Strong staining appears in adipocytes or cardiomyocytes. | Those cells are reported as not detected (HPA tissue IHC). Check a matched negative control and the stained cell identity; cross-reactivity or endogenous chromogenic activity are possible explanations (standard IHC practice). |
| Broad, diffuse color covers cells and surrounding tissue without clear cell boundaries. | This obscures the cell-specific cytoplasmic pattern used for interpretation (HPA tissue IHC). Compare a negative control, then assess blocking, washing and detection background (standard IHC practice). |
| A known-positive cell population has no visible signal. | Absence in a reported high population, such as colon glandular cells, calls for a staining-run check (HPA tissue IHC). Review antibody dilution, detection and retrieval conditions as general IHC variables (standard IHC practice). |
💡Expected MANF appearanceA convincing positive is cell-associated cytoplasmic staining in a reported high population, such as bone marrow hematopoietic cells (HPA tissue IHC); nuclear-dominant color or comparable signal in negative controls argues against a specific result (HPA ICC-IF; standard IHC practice).
| Intracellular retention and secretion | MANF resides in the ER or sarcoplasmic reticulum under normal conditions but can be secreted with ER stress or hypoxia (UniProt P55145). Interpret extracellular color cautiously; its presence alone does not establish cell of origin. |
| Cell-specific tissue distribution | HPA reports high staining in selected immune, neuronal and glandular cells, while adipocytes and cardiomyocytes are not detected (HPA tissue IHC). Score the identified cell population, not the tissue as a whole. |
| Antibody evidence | The listed HPA011175 rabbit polyclonal antibody is Approved for IHC and ICC; the tissue profile has medium RNA–protein consistency (HPA antibodies; HPA tissue IHC). Treat unexpected staining as requiring controls, not as a new expression claim. |
| Protein processing | UniProt lists a signal peptide at residues 1–24 and a mature chain at 25–182 (UniProt P55145). The supplied evidence gives no antibody epitope, so processing cannot predict this antibody’s staining strength. |
| Subcellular comparison | HPA ICC-IF supports ER localization, consistent with UniProt’s ER-lumen assignment (HPA ICC-IF; UniProt P55145). This supports compartment interpretation; ICC-IF images do not specify an IHC paraffin-section protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in colon glandular cells. | Colon glandular cells are reported high, so a blank result may reflect a failed IHC step (HPA tissue IHC; standard IHC practice). | Run a reported-positive control alongside the sample; check antibody dilution, retrieval, detection reagents and timing against the assay instructions (standard IHC practice). |
| Every compartment shows brown color. | Widespread color is hard to reconcile with cell-specific cytoplasmic MANF staining (HPA tissue IHC); detection background is possible (standard IHC practice). | Compare the negative control and inspect blocking, washing and chromogen development; adjust only the step implicated by the control (standard IHC practice). |
| Signal is mainly nuclear. | The reported tissue pattern is cytoplasmic, and the supported ICC-IF location is ER (HPA tissue IHC; HPA ICC-IF). | Confirm compartment boundaries with the counterstain and compare a matched negative control before calling the nuclear signal specific (standard IHC practice). |
| Adipocytes or cardiomyocytes stain strongly. | Both cell types are listed as not detected in HPA tissue IHC; nonspecific staining or endogenous detection activity is possible (HPA tissue IHC; standard IHC practice). | Verify cell identity and review negative controls and detection blocking before interpreting the signal as MANF (standard IHC practice). |
| Staining differs between otherwise comparable specimens. | MANF can be up-regulated and secreted during ER stress or hypoxia (UniProt P55145); that biology alone cannot explain a particular slide difference. | First compare cell types and matched staining controls; document specimen context before assigning a biological explanation (HPA tissue IHC; standard IHC practice). |
| Can an ICC-IF image define the expected IHC result? | HPA ICC-IF supports ER localization, while HPA tissue IHC reports cell-specific cytoplasmic staining (HPA ICC-IF; HPA tissue IHC). | Use ICC-IF as a compartment cross-check; judge paraffin-section staining against the tissue IHC pattern and its controls (HPA tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing MANF staining
A03825 · IHCImmunohistochemical analysis of paraffin-embedded rat-brain, antibody was diluted at 1:100
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000145050-MANF/tissue/cerebral+cortex#imid_3054392
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000145050-MANF/tissue/colon#imid_3054318
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000145050-MANF/tissue/liver#imid_3054327
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000145050-MANF/tissue/pancreas#imid_3054262
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA011175; green = MANF, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA011175; green = MANF, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MANF IHC & IF
🧪Run colon first: glandular cells should stain strongly (HPA: High in colon glandular cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the colon slide, compare glandular staining with adjacent cells that remain at background, without assuming those cells are biologically MANF-negative (HPA: colon glandular-cell result only).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show MANF in A-431, U-251MG, U2OS, with annotated localisation: Endoplasmic reticulum (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; use host- and isotype-matched irrelevant IgG for a monoclonal primary or host-matched nonimmune IgG for a polyclonal primary; and confirm specificity with MANF knockout material or an immunogen-peptide block where available (standard IHC controls). For chromogenic colon IHC, block endogenous peroxidase and check for endogenous biotin if using avidin–biotin detection (standard IHC practice).
⚠️Feasibility: The supplied evidence reports no MANF-specific fixation window or antigen-retrieval dependence; test retrieval conditions empirically (supplied target/application evidence; standard IHC practice). The selected rat-brain paraffin-section caption reports a 1:100 dilution but leaves the fixative unreported; it does not establish fixation (selected IHC caption: A03825). Frozen sections and IF are not established as easier by these data; in colon, assess glandular staining against luminal background, and interpret any intracellular ER-like signal alongside MANF’s capacity for secretion under stress (HPA: High in colon glandular cells; HPA: ER localization in ICC-IF; UniProt P55145: stress-induced secretion). The selected A03825 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image A03825).
HPA tissue IHC evidence for MANF
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Secreted protein, tissue location of RNA and protein is expected to differ.). 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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | 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 → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Vagina | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MANF IHC Tips
Troubleshoot MANF staining in paraffin sections by checking retrieval, controls, cellular location, and scoring before interpreting changes in chromogenic signal.
How should I retrieve MANF in paraffin sections when staining is weak?
Use citrate buffer at pH 6.0 for heat-induced retrieval at 95–98 °C for 20 minutes (page retrieval setting). Let sections cool consistently, then compare retrieved and untreated serial sections with the same antibody dilution and detection conditions (standard IHC practice). A paraffin-embedded rat-brain image used the catalog antibody at 1:100, but its caption does not report retrieval conditions (A03825 tissue-IHC caption). If staining remains weak, test a different retrieval buffer or pH on adjacent sections as a documented fallback, while watching for tissue damage and increased background (standard IHC practice).
Could fixation explain inconsistent MANF staining between paraffin specimens?
MANF-specific fixation sensitivity is unknown here because the selected rat-brain paraffin caption does not state a fixative (A03825 tissue-IHC caption). Record fixative, fixation duration, processing schedule, and section age for each specimen before comparing staining intensity (standard IHC practice). When possible, stain serial sections from specimens processed together in one run, with the same retrieval and detection settings (standard IHC practice). Compare a range of documented fixation durations on matched material if inconsistent signal persists; keep the tissue and scoring method constant (standard IHC practice). Do not assign a weak MANF result to fixation alone without that comparison (standard IHC practice).
Which staining pattern is plausible for MANF in tissue sections?
Expect predominantly cytoplasmic staining compatible with the endoplasmic reticulum, rather than a required nuclear pattern (HPA tissue IHC profile; HPA subcellular: endoplasmic reticulum). MANF occupies the ER lumen under normal conditions and can be secreted during ER stress or hypoxia (UniProt P55145 subcellular annotation). Its sequence has no transmembrane segment, so a crisp membrane rim is not an expected default pattern (UniProt P55145 topology). Assess cellular and possible extracellular signal separately, because a secreted protein can appear away from its site of synthesis (UniProt P55145 subcellular annotation; HPA reliability description). Check compartment patterns against a positive control on the same staining run (standard IHC practice).
How do MANF processing and epitope location affect IHC interpretation?
MANF has an annotated signal peptide at residues 1–24 and a mature chain at residues 25–182 (UniProt P55145 processing). No isoforms or glycosylation sites are annotated, while phosphotyrosine is noted at residue 76 (UniProt P55145 isoforms, glycosylation and modified residues). The supplied antibody evidence does not map its epitope, so staining cannot establish whether the reagent recognizes precursor, mature MANF, or a modification-dependent site (provided antibody evidence). If results conflict across antibodies, compare their stated immunogens where available and stain matched serial sections under identical retrieval conditions (standard IHC practice). Interpret agreement by cellular compartment and controls, not intensity alone (standard IHC practice).
How can IF help assess MANF-positive cells and subcellular signal?
For a separate IF experiment, pair MANF with a marker for the expected cell type, such as a neuronal marker when examining caudate neurons (HPA tissue IHC: high in caudate neuronal cells; standard IF practice). Choose fluorophores after inspecting unstained tissue for autofluorescence, and keep emission channels sufficiently separated for multiplex imaging (standard IF practice). Because MANF is reported in the ER lumen, use a permeabilisation condition that permits antibody access to intracellular compartments, then compare with an appropriate control (UniProt P55145 subcellular annotation; standard IF practice). An ER marker can help assess the reported ER location, while separate channels help identify bleed-through (HPA subcellular: endoplasmic reticulum; standard IF practice).
What should I check when MANF chromogenic staining looks diffuse?
Compare the section with a no-primary control to identify signal from the detection system, and inspect tissue edges and damaged regions separately (standard IHC practice). Include a peroxidase block before a peroxidase-based DAB detection step; those are general chromogenic workflow measures, not MANF-specific validation (standard IHC practice). The catalog antibody's paraffin rat-brain image reports a 1:100 dilution, which is a documented comparison point for that specimen rather than a universal optimum (A03825 tissue-IHC caption). Titrate antibody concentration and detection time on adjacent sections while holding retrieval constant (standard IHC practice). Judge diffuse signal against the expected cytoplasmic pattern (HPA tissue IHC profile).
How should I quantify MANF IHC across tissue groups? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis region before scoring, then use the same exposure-independent brightfield acquisition and threshold rules across slides (standard IHC practice). For cellular staining, report percent positive cells and an H-score from 0–300, with intensity categories applied consistently (standard IHC scoring practice). If cells are sparse, report positive-cell density per mm² alongside the number of eligible cells counted (standard IHC scoring practice). Normalize comparisons to the same cell type, tissue area, and staining run, using a shared control section to monitor batch variation (standard IHC practice). Score extracellular staining separately from cellular staining because MANF can be secreted (UniProt P55145 subcellular annotation).
How can I distinguish genuine MANF staining from artefact?
Give greatest weight to reproducible cytoplasmic staining in plausible cells, supported by a clean no-primary control and consistent serial sections (HPA tissue IHC profile; standard IHC practice). HPA reports high staining in caudate neurons and bone-marrow hematopoietic cells, providing context for those tissues (HPA tissue IHC). A strong nuclear-only or sharply membrane-bound pattern warrants scrutiny against MANF's ER-lumen location and lack of a transmembrane segment (UniProt P55145 subcellular annotation and topology). Exclude edge staining, necrotic regions, and residual endogenous peroxidase signal before scoring DAB deposits (standard IHC practice). Interpret extracellular signal cautiously because MANF can be secreted during ER stress or hypoxia (UniProt P55145 subcellular annotation).
Boster reagents
Best MANF / Mesencephalic astrocyte-derived neurotrophic factor IHC Antibodies
The catalog includes an anti-MANF IHC image from paraffin-embedded rat brain (A03825 image caption), listed IF use (A03825 applications), and human, mouse, and rat reactivity (catalog reactivity).
Real IHC data
Anti-ARP Antibody
Cat # A03825
The rendered card is A03825, whose IHC image shows paraffin-embedded rat brain at 1:100 (A03825 image caption). A03825 lists IHC and IF applications and human, mouse, and rat reactivity; no IF image is provided (A03825 applications/reactivity/image alts).
Which to pick: For tissue IHC, pick A03825: its own image documents paraffin-embedded rat brain, with the fixative unreported (A03825 image caption). For IF/ICC, A03825 lists IF at 1:50, but ICC validation and an IF image are unreported (A03825 applications/dilution/image alts). For work across human, mouse, and rat, pick A03825 for its broader listed IHC and IF applications and documented polyclonal format; A03825-2 lists the same species but IHC without IF (catalog reactivity/applications; A03825 dilution raw).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P55145 (MANF_HUMAN, Mesencephalic astrocyte-derived neurotrophic factor).
- Human Protein Atlas. MANF tissue IHC expression (reliability: Approved).
- Human Protein Atlas. MANF subcellular location (ICC-IF): Localized to the endoplasmic reticulum..
- Human Protein Atlas. MANF antibody validation summary (1 antibodies).
- MANF Promotes Unexplained Recurrent Miscarriages by Interacting with NPM1 and Downregulating Trophoblast Cell Migration and Invasion. International journal of biological sciences 2024 — PMC10750294.
- Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Is Highly Expressed in Mouse Tissues With Metabolic Function. Frontiers in endocrinology 2019 — PMC6851024.
- Altered MANF Expression in Pancreatic Acinar and Ductal Cells in Chronic Alcoholic Pancreatitis: A Cross-Sectional Study. Biomedicines 2023 — PMC9953319.
- Spatiotemporal expression of MANF in the developing rat brain. PloS one 2014 — PMC3938758.
- PubMed PMID:8649854 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:16641997 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MANF (Mesencephalic astrocyte-derived neurotrophic factor). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); ER lumen (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A03825 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded rat brain tissue; fixative not specified (datasheet A03825); 2. Fixation: Image fixative and duration unreported (datasheet A03825); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-MANF, 1:100-1:300 (datasheet A03825); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03825); Caveat: Secretion may separate protein staining from RNA expression (HPA tissue IHC); Isoform / epitope: No annotated isoforms; mature chain 25–182 has no membrane span (UniProt); Regulation: ER stress or hypoxia up-regulates MANF (UniProt). Evidence: UniProt P55145, HPA tissue IHC · Approved, HPA ICC-IF · endoplasmic reticulum., Datasheet A03825, PMC10750294, PMC6851024. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MANF/immunohistochemistry-ihc-guide.