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MLF2 / Myeloid leukemia factor 2 · IHC design guide
Design Immunohistochemistry for MLF2
Plan paraffin-section MLF2 IHC around cytoplasmic and nuclear staining (HPA tissue IHC). Start the IHC-validated antibody at 1:100–1:200 (datasheet), using high-staining bone marrow hematopoietic cells and low-staining smooth muscle cells to assess the expected range (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MLF2 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 and nuclear staining (HPA tissue IHC) | |
| Staining pattern | General cytoplasmic and nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A12895) | |
| Caveat | Smooth muscle cells show low staining (HPA tissue IHC) | |
| Regulation | Regulation unreported (UniProt) | |
| Isoform / epitope | No isoforms annotated; chain 1–248 (UniProt) |
Section 1
Recommended MLF2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by 1 published mouse spinal tissue protocol (PMC5508040).
| Sample | Paraffin-embedded rat liver tissue; fixative not specified (datasheet A12895) |
| Fixation | Image fixative and duration unreported (datasheet A12895); 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 Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear 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-MLF2, 1:100-1:200 (datasheet A12895) |
| 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 | MLF2-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic and nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min (page antigen-retrieval rule); consider the published citrate-based treatment for mouse spinal tissue (PMC5508040).
Section 2
What Is the Expected MLF2 Staining Pattern?
In paraffin-section IHC, expect MLF2 in cytoplasm and nuclei across many cell types (HPA: general cytoplasmic and nuclear expression; UniProt Q15773: ubiquitous expression). HPA reports high staining in selected epithelial, hematopoietic, and neuronal cells and low staining in smooth muscle cells (HPA: tissue IHC). Its annotated protein has no transmembrane segment (UniProt Q15773: topology). The tissue IHC profile has Enhanced reliability (HPA: tissue IHC).
| Cytoplasmic and nuclear chromogen in the expected cells, with identifiable cell boundaries and nuclei. | This fits the reported tissue pattern (HPA: general cytoplasmic and nuclear expression). Judge the compartments within each cell type rather than expecting identical intensity across a section; HPA reports both high and low cell-type staining (HPA: tissue IHC). |
| Signal is confined to extracellular material or an epithelial lumen, with little cellular cytoplasmic or nuclear staining. | That distribution conflicts with the reported IHC pattern (HPA: general cytoplasmic and nuclear expression). Treat it as a possible staining artefact and check morphology and detection controls; a membrane-like IF signal alone does not establish a luminal IHC pattern (HPA: subcellular ICC-IF). |
| Strong staining appears in an unexpected cell population while expected cells are weak or unstained. | Possible causes include cross-reactivity or endogenous detection activity (general IHC practice). Compare the cell types carefully: smooth muscle is reported as low, not negative, and HPA supplies no negative tissue for this profile (HPA: tissue IHC). |
| A uniform chromogenic haze covers tissue and blank spaces, obscuring nuclei and cytoplasm. | This does not resolve the reported cellular pattern (HPA: tissue IHC). Background from the detection workflow is a possibility (general IHC practice); compare a control omitting primary antibody before assigning the haze to MLF2. |
| No cellular signal appears in a section expected to contain high-staining cells. | The run may have failed if appropriate high-staining cells are present (HPA: tissue IHC). First confirm cell identity and preservation, then review the IHC-P staining and detection steps (general IHC practice); one blank section cannot establish absent MLF2. |
💡Expected MLF2 appearanceCall a result positive when the expected cells show discernible cytoplasmic and nuclear staining, potentially high in HPA-listed cell types; isolated extracellular deposit or diffuse haze is a suspect positive (HPA: tissue IHC; general IHC practice).
| Tissue and cell type | High staining is reported in adrenal and appendix glandular cells, bone marrow hematopoietic cells, and cortical neuronal cells; smooth muscle cells are low (HPA: tissue IHC). Select and score the relevant cell population. |
| Compartment evidence | Tissue IHC shows general cytoplasmic and nuclear expression (HPA: tissue IHC). ICC-IF places MLF2 mainly in nucleoplasm, with additional cytosol and plasma membrane localization (HPA: subcellular ICC-IF). Interpret each assay against its own evidence. |
| IHC validation | The tissue profile has Enhanced reliability, described as high consistency between antibody staining and RNA expression (HPA: tissue IHC). HPA010811 and HPA010859 each have Enhanced IHC status (HPA: antibodies). Validation supports the reported pattern, not every deposit on an individual slide. |
| Protein annotation | UniProt annotates cytoplasmic and nuclear localization, no transmembrane segment, no signal peptide, and a single chain spanning residues 1–248 (UniProt Q15773). These annotations do not establish a target-specific retrieval condition or fixation sensitivity. |
| IF/ICC Q&A | Where should IF/ICC signal appear? Mainly in nucleoplasm, with additional cytosol and plasma membrane signal (HPA: subcellular ICC-IF). That is the IF/ICC localization report; use the tissue IHC profile to assess paraffin-section chromogen (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known high-staining cell types show no signal. | The expected cells may be absent from the section, or an IHC-P workflow step may have failed (HPA: tissue IHC; general IHC practice). | Confirm the cell type on the counterstained slide; run a high-staining reference tissue and review the antibody, retrieval, and detection steps using their documented IHC-P instructions (general IHC practice). |
| Only extracellular or luminal material stains. | The location conflicts with general cytoplasmic and nuclear tissue staining (HPA: tissue IHC); deposited chromogen or nonspecific detection is possible (general IHC practice). | Inspect morphology and compare a primary-omission control; score MLF2 only where cellular compartments can be identified (general IHC practice; HPA: tissue IHC). |
| The whole section has diffuse background. | Detection-system background can obscure a cellular pattern (general IHC practice). HPA's Enhanced designation does not validate this individual run (HPA: tissue IHC). | Compare a primary-omission control and review blocking, washing, and chromogen development against the established IHC-P workflow (general IHC practice). |
| Smooth muscle is weaker than adjacent cell populations. | Low smooth-muscle-cell staining is reported (HPA: tissue IHC); weakness there alone does not indicate failed detection. | Score smooth muscle separately and check a reported high-staining cell population on a suitable section (HPA: tissue IHC). |
| A bright signal appears in an unexpected cell type. | Cross-reactivity or endogenous detection activity is possible (general IHC practice); HPA lists no negative tissue for this profile (HPA: tissue IHC). | Check cell identity and localization, then compare detection controls and the reported tissue pattern before calling the signal MLF2 (general IHC practice; HPA: tissue IHC). |
| An IF/ICC image looks more nuclear than the IHC section. | ICC-IF reports mainly nucleoplasmic localization, while tissue IHC reports general nuclear and cytoplasmic expression (HPA: subcellular ICC-IF; HPA: tissue IHC). | Assess each image using its assay-specific localization report; do not use the IF/ICC plasma membrane finding as an IHC-P scoring requirement (HPA: subcellular ICC-IF; HPA: tissue IHC). |
Experiment evidence
IHC & IF images showing MLF2 staining
A12895 · IHCImmunohistochemistry of paraffin-embedded rat liver using MLF2 antibody at dilution of 1:100 .Perform microwave antigen retrieval with 10 mM
A12895 · IHCImmunohistochemistry of paraffin-embedded human esophageal using MLF2 antibody at dilution of 1:100 .Perform microwave antigen retrieval wit
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000089693-MLF2/tissue/cerebral+cortex#imid_2890364
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000089693-MLF2/tissue/colon#imid_2890298
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000089693-MLF2/tissue/liver#imid_2890291
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000089693-MLF2/tissue/kidney#imid_2890284
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA010811; green = MLF2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA010811; green = MLF2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MLF2 IHC & IF
🧪Run appendix first and look for staining in glandular cells (HPA: High in appendix glandular cells). HPA detects MLF2 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and treat any unstained non-glandular cells on the slide as background references rather than validated negative cells (HPA: no negative rows).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: MLF2 is detected in all 45 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 MLF2 in A-431, U-251MG, U2OS, NIH 3T3, with annotated localisation: Nucleoplasm (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, an immunoglobulin control matched to the primary antibody’s host species and class, and a knockout specimen or peptide-block control if available (standard IHC practice). Quench endogenous peroxidase and check inflammatory cells in appendix for residual chromogen signal (standard chromogenic IHC practice).
⚠️Feasibility: No MLF2-specific fixation window or fixation effect is reported in the supplied evidence; the selected A12895 paraffin-section caption does not state a fixative (A12895 caption: rat liver paraffin section; fixative unreported). That caption specifies microwave retrieval in 10 mM PBS, pH 7.2, but does not establish that retrieval is required for every specimen (A12895 caption). The evidence does not establish whether frozen sections or IF are easier than paraffin IHC; appendix inflammatory cells warrant attention to endogenous peroxidase (standard chromogenic IHC practice).
HPA tissue IHC evidence for MLF2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: MLF2 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced MLF2 IHC Tips
Troubleshoot MLF2 staining in paraffin sections by checking retrieval, compartment-specific signal, controls and scoring before interpreting chromogenic IHC.
How should I troubleshoot weak MLF2 staining after antigen retrieval?
Start with Tris-EDTA pH 9.0 heat-induced retrieval at 95–98 °C for 20 min (page retrieval setting). If staining remains weak, test microwave retrieval in 10 mM PBS at pH 7.2 on adjacent sections; that condition was used for the catalog antibody in paraffin-embedded rat liver (A12895 image caption). Keep antibody dilution, detection and imaging settings constant across the comparison so a stronger signal can be attributed to retrieval (standard IHC practice). Inspect tissue morphology and background alongside nuclear and cytoplasmic staining, since both compartments are plausible for MLF2 (UniProt Q15773 subcellular annotation; HPA tissue IHC profile).
Could fixation explain inconsistent MLF2 staining across paraffin blocks?
Target-specific sensitivity to fixative choice or fixation duration is unknown; the selected paraffin-section caption does not state a fixative (A12895 image caption). Record the fixative and processing history for each block, then compare sections with similar handling and thickness under the same retrieval and detection conditions (standard IHC practice). If a controlled comparison is possible, assess signal intensity and tissue morphology together rather than treating loss of staining as proof of MLF2 loss (standard IHC practice). The reported nuclear and cytoplasmic distribution can guide compartment checks, but it does not establish a fixation requirement (UniProt Q15773 subcellular annotation; HPA tissue IHC profile).
Which MLF2 staining compartments should I accept in chromogenic IHC?
Assess nuclear and cytoplasmic staining separately because both are reported for MLF2 in tissue, and UniProt also lists both compartments (HPA tissue IHC profile; UniProt Q15773 subcellular annotation). HPA subcellular imaging places MLF2 mainly in the nucleoplasm, with additional cytosol and plasma-membrane localisation (HPA subcellular summary). A membrane rim without convincing intracellular signal deserves scrutiny, especially when adjacent cells share the same edge staining (HPA subcellular summary; standard IHC practice). Compare the pattern with a matched control section and the expected cell population; HPA reports high staining in adrenal glandular cells and low staining in smooth muscle cells (HPA tissue IHC).
How can I assess whether an MLF2 epitope is accessible in sections?
Check the antibody's documented immunogen or epitope before interpreting a negative section; the supplied record does not identify the catalog antibody's epitope (supplied product evidence). UniProt lists one 248-aa chain, no annotated isoforms, no transmembrane segment and no annotated domains, so these annotations do not identify a section-specific epitope barrier (UniProt Q15773). Phosphoserines at positions 217, 238 and 240 are annotated, but their effect on this antibody is unknown without epitope information (UniProt Q15773 modified residues). Compare retrieval conditions on adjacent sections while holding detection constant, and judge both signal and morphology before assigning an epitope-related cause (standard IHC practice).
How should I plan a companion MLF2 immunofluorescence experiment?
Use an epithelial marker to identify glandular cells when examining an appropriate tissue, since HPA reports high MLF2 staining in adrenal glandular cells (HPA tissue IHC; standard IF practice). Select spectrally separated fluorophores and place the weaker signal in a channel with low tissue autofluorescence, checking an unstained section first (standard IF practice). For intracellular MLF2, optimise permeabilisation to expose nuclear and cytosolic epitopes while preserving morphology; UniProt reports no transmembrane segment (UniProt Q15773 topology; HPA subcellular summary; standard IF practice). Validate the IF antibody and fixation conditions independently: the catalog image documents paraffin-section chromogenic IHC, and its fixative is unreported (A12895 image caption).
What should I check when MLF2 IHC has diffuse brown background?
Compare a no-primary control with the stained section to distinguish detection-system staining from antibody-dependent signal (standard IHC practice). For peroxidase-based chromogenic detection, check the peroxidase block, antibody dilution, washes and DAB development time on matched sections; these are general workflow variables (standard IHC practice). The catalog image used antibody at 1:100 in paraffin-embedded rat liver, which is a documented starting point for that image rather than a universal dilution (A12895 image caption). Treat diffuse haze cautiously because MLF2 can appear in both nucleus and cytoplasm; require cell-shaped staining above the control background (HPA tissue IHC profile; standard IHC practice).
How should I score heterogeneous MLF2 staining in tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and score nuclear and cytoplasmic compartments separately, since MLF2 staining is reported in both (HPA tissue IHC profile; UniProt Q15773 subcellular annotation). Record the percentage of positive cells and intensity grades, then calculate an H-score from 0–300 if graded intensity is reproducible (standard IHC practice). For spatial counts, report positive cells per mm² of viable tissue and normalise to the number or area of eligible cells, rather than total section area (standard IHC practice). Apply the same threshold, exposure-independent chromogenic analysis settings and control criteria to every section, and record excluded necrotic or damaged regions (standard IHC practice).
How can I distinguish true MLF2 positivity from staining artefact?
Look for reproducible nuclear or cytoplasmic staining in intact cells; both compartments are supported, while HPA describes mainly nucleoplasmic localisation in subcellular imaging (UniProt Q15773 subcellular annotation; HPA tissue IHC profile; HPA subcellular summary). Check whether the stained cells fit the sampled tissue: HPA reports high staining in adrenal glandular cells and low staining in smooth muscle cells (HPA tissue IHC). Question signal confined to section edges, necrotic areas or a no-primary control, and check endogenous peroxidase when using DAB (standard IHC practice). Resolve an unexpected membrane-only pattern with matched controls and independent antibody validation before calling it MLF2 (HPA subcellular summary; standard IHC practice).
Boster reagents
Best MLF2 / Myeloid leukemia factor 2 IHC Antibodies
A12895 has paraffin-section IHC images from rat liver and human esophageal tissue (catalog image captions); no IF image is supplied (catalog image list).
Real IHC data
Anti-Myeloid leukemia factor 2 MLF2 Antibody
Cat # A12895
A12895 is listed for IHC and reacts with human, mouse and rat (catalog applications and reactivity). Its IHC images show rat liver and human esophageal paraffin sections at 1:100 after microwave retrieval in 10 mM PBS, pH 7.2 (A12895 image captions).
Which to pick: Choose A12895 for paraffin-section tissue IHC, supported by its own rat liver and human esophageal images; the fixative is unreported (A12895 image captions). For IF/ICC, A12895 has no supplied IF image or IF dilution, so an IF recommendation cannot be validated from these data (catalog IF fields). For cross-species IHC, A12895 lists human, mouse and rat reactivity, with image evidence for human and rat only (catalog reactivity; A12895 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q15773 (MLF2_HUMAN, Myeloid leukemia factor 2).
- Human Protein Atlas. MLF2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. MLF2 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the plasma membrane and cytosol..
- Human Protein Atlas. MLF2 antibody validation summary (2 antibodies).
- Spinal poly-GA inclusions in a C9orf72 mouse model trigger motor deficits and inflammation without neuron loss. Acta neuropathologica 2017 — PMC5508040.
- PubMed PMID:8661158 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:17081983 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MLF2 (Myeloid leukemia factor 2). Expected localisation Cytoplasmic and nuclear staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A12895 at 1:100-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded rat liver tissue; fixative not specified (datasheet A12895); 2. Fixation: Image fixative and duration unreported (datasheet A12895); 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 Tris-EDTA buffer, pH 9.0 , 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-MLF2, 1:100-1:200 (datasheet A12895); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A12895); Caveat: Smooth muscle cells show low staining (HPA tissue IHC); Isoform / epitope: No isoforms annotated; chain 1–248 (UniProt); Regulation: Regulation unreported (UniProt). Evidence: UniProt Q15773, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A12895, PMC5508040. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MLF2/immunohistochemistry-ihc-guide.