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MTHFD2 / Bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase, mitochondrial · IHC design guide
Design Immunohistochemistry for MTHFD2
Plan chromogenic MTHFD2 IHC in paraffin sections with the IHC-validated antibody A06465-1 (datasheet: IHC). Compare cytoplasmic staining in kidney tubule cells with the undetected signal reported for adipocytes (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MTHFD2 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); mitochondrial protein (UniProt) | |
| Staining pattern | Cytoplasmic signal in several tissues, distinct in immune cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A06465-1) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across compared paraffin sections. (selected-SKU IHC image A06465-1) | |
| Caveat | Immune-cell staining can complicate mixed-tissue scoring (HPA tissue IHC) | |
| Regulation | Staining intensity varies by cell type (HPA tissue IHC) | |
| Isoform / epitope | 2 isoforms; mature chain 36–350; epitope map unspecified (UniProt) |
Section 1
Recommended MTHFD2 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet A06465-1) with published MTHFD2 IHC protocols for esophageal and lung cancer tissues (PMC7055195; PMC12009792; PMC6991687).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A06465-1) |
| Fixation | Image fixative and duration unreported (datasheet A06465-1); 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 A06465-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A06465-1) |
| Primary antibody | Rabbit anti-MTHFD2, 1:50 recommended; image 1:100 (datasheet A06465-1) |
| Primary incubation | Overnight at 4 °C (datasheet A06465-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A06465-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | MTHFD2-positive staining in lymphoid tissue of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several different tissues, distinct expression in immune cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet A06465-1). The published citrate and low-pH conditions used other antibodies (PMC7055195; PMC12009792).
Section 2
What Is the Expected MTHFD2 Staining Pattern?
MTHFD2 is mitochondrial and has no transmembrane segment (UniProt P13995 topology). In paraffin sections, expect cytoplasmic staining in selected cells, including kidney tubules, intestinal glands, and lymph node germinal centers (HPA tissue IHC). HPA rates its tissue staining Approved, with medium agreement between staining and RNA data and external verification pending (HPA tissue IHC). Interpret weak or unexpected staining with that limitation in mind.
| Cytoplasmic staining in kidney tubular cells, intestinal glandular cells, or lymph node germinal center cells. | This matches cell populations scored High by HPA tissue IHC. Mitochondrial localization supports a cytoplasmic location (UniProt P13995; HPA subcellular ICC-IF). A chromogenic section need not resolve individual mitochondria; assess the stained cells and compartment together. |
| Predominantly nuclear or cell-surface staining, with little corresponding cytoplasmic signal. | That distribution conflicts with mitochondrial localization (UniProt P13995; HPA subcellular ICC-IF). Treat it as a possible staining artefact or off-target signal, especially if the expected cytoplasmic pattern is absent; localization alone cannot identify its cause. |
| Strong staining in adipocytes or cardiomyocytes, particularly without staining in an expected positive cell population. | HPA reports MTHFD2 as Not detected in adipocytes of adipose tissue and cardiomyocytes of heart muscle (HPA tissue IHC). Investigate possible cross-reactivity or endogenous detection activity before calling these cells positive. The HPA result is a comparison point, not proof that every specimen must be negative. |
| Broad, even color across cells and surrounding tissue, obscuring cell boundaries. | This does not resemble HPA's cell-specific cytoplasmic pattern (HPA tissue IHC). Consider nonspecific antibody binding, incomplete blocking, or detection background as general IHC possibilities. Check a control without primary antibody and compare staining across the expected positive and negative cell populations. |
| No visible signal in kidney tubular cells or lymph node germinal center cells. | Both are scored High in HPA tissue IHC, so their absence calls for a technical check. Confirm that the section contains the named cells, then review the antibody's validated IHC-P conditions and detection controls (general IHC practice). A single unstained section does not establish biological absence. |
💡Expected MTHFD2 appearanceCall a result positive when identifiable HPA High cell populations show cytoplasmic chromogenic staining; predominantly nuclear staining or strong color in HPA Not detected cells warrants investigation (HPA tissue IHC; UniProt P13995; HPA subcellular ICC-IF).
| Compartment and optical resolution | MTHFD2 is mitochondrial and lacks a transmembrane segment (UniProt P13995 topology). HPA tissue IHC describes cytoplasmic expression, while HPA ICC-IF supports mitochondrial localization. Score the cytoplasmic pattern in IHC without requiring visible mitochondrial puncta. |
| Cell population sampled | HPA scores duodenal and small-intestinal glandular cells, kidney tubular cells, and tonsil germinal center cells High; lung alveolar cells are Low (HPA tissue IHC). Name the cell population when interpreting a section, since a tissue-wide positive or negative label would hide that difference. |
| Strength of validation | The tissue IHC profile is Approved but has medium staining-to-RNA agreement and awaits external verification (HPA tissue IHC). HPA lists two antibodies with Approved IHC status, HPA049657 and CAB003684 (HPA antibodies). These ratings support comparison with the reported pattern without guaranteeing every specimen will match. |
| Isoforms and processing | UniProt lists two isoforms and a mature chain spanning residues 36–350 (UniProt P13995). No antibody epitope is supplied, so this record cannot establish whether either isoform or the processed region changes the observed IHC pattern. |
| Chromogenic detection | Endogenous enzyme activity can produce color when the matching enzyme-based detection system is used (general IHC practice). A control without primary antibody helps distinguish detection background from antibody-dependent staining; this is a workflow check, not an MTHFD2-specific effect. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are unstained. | The named cells may be absent from the section, or the staining run may have failed (general IHC practice). | Confirm the cell population on the counterstained section; include a tissue and cell population scored High by HPA, then check the antibody's validated IHC-P conditions and run controls (HPA tissue IHC; general IHC practice). |
| Signal is mainly nuclear or along cell borders. | The compartment conflicts with mitochondrial MTHFD2 localization; artefact or off-target binding is possible (UniProt P13995; HPA subcellular ICC-IF). | Compare with a control lacking primary antibody and with a known positive cell population. Reassess specificity before scoring the unusual compartment as MTHFD2 (general IHC practice). |
| Adipocytes or cardiomyocytes stain strongly. | Those cell populations are reported Not detected (HPA tissue IHC); cross-reactivity or detection background is possible. | Inspect a control without primary antibody and compare the signal with HPA High cell populations on appropriately stained sections. Record the cell type and compartment rather than assigning positivity from color alone (HPA tissue IHC; general IHC practice). |
| Diffuse color prevents cell-by-cell scoring. | Nonspecific binding or background from the detection system may obscure the cell-specific pattern (general IHC practice; HPA tissue IHC). | Review blocking and antibody use against the validated IHC-P procedure; check a control without primary antibody. Score only when cytoplasmic signal can be assigned to identifiable cells (general IHC practice). |
| A negative comparison looks as dark as the expected positive cells. | The contrast does not follow HPA's reported High versus Not detected cell populations; background or an unsuitable comparison is possible (HPA tissue IHC). | Verify the exact cell types being compared and examine detection controls. Interpret differences cautiously because HPA rates tissue IHC agreement as medium and external verification is pending (HPA tissue IHC). |
| What should an IF/ICC image show? | HPA reports a supported mitochondrial location in ICC-IF, whereas its tissue IHC summary describes cytoplasmic staining (HPA subcellular ICC-IF; HPA tissue IHC). | For IF/ICC interpretation, look for mitochondrial localization and use the separate IF/ICC guide for experimental conditions. Do not require resolved mitochondrial puncta to score a chromogenic paraffin section (HPA subcellular ICC-IF; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing MTHFD2 staining
A06465-1 · IHCIHC analysis of MTHFD2 using anti-MTHFD2 antibody (A06465-1). MTHFD2 was detected in a paraffin-embedded section of human colon cancer tissu
A06465-1 · IHCIHC analysis of MTHFD2 using anti-MTHFD2 antibody (A06465-1). MTHFD2 was detected in a paraffin-embedded section of human lung cancer tissue
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000065911-MTHFD2/tissue/cerebral+cortex#imid_15287970
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000065911-MTHFD2/tissue/colon#imid_15287888
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000065911-MTHFD2/tissue/liver#imid_15287889
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000065911-MTHFD2/tissue/kidney#imid_15287882
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA049657; green = MTHFD2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA049657; green = MTHFD2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MTHFD2 IHC & IF
🧪Run kidney first: tubular cells should stain (HPA: High in kidney cells in tubules). Use adipose tissue as the negative comparator: adipocytes are listed as not detected (HPA: Not detected in adipocytes); on the kidney slide, any non-tubular cells selected as internal negatives should show background signal only, although their negative status is not established by the supplied HPA row.
Positive control tissue: Appendix (Lymphoid tissue, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show MTHFD2 in A-431, U-251MG, U2OS, NIH 3T3, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host-matched rabbit isotype controls, matching antibody class and concentration where possible; the primary’s clonality is unreported (caption: rabbit anti-MTHFD2). A matched MTHFD2 knockout specimen would provide a biological negative; quench endogenous peroxidase for HRP/DAB detection and account for endogenous biotin in kidney tubules if using avidin–biotin detection (caption: HRP/DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A06465-1 paraffin-section caption does not state a fixative (caption: fixative not stated). That caption uses heat retrieval in EDTA at pH 8.0, so retrieval is documented for its IHC result, but whether MTHFD2 staining depends on it is unreported (caption: EDTA retrieval). The supplied evidence does not establish that frozen sections or IF are easier; kidney tubules can present endogenous biotin artefact with avidin–biotin detection (standard IHC practice).
HPA tissue IHC evidence for MTHFD2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Lymphoid tissue | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MTHFD2 IHC Tips
Troubleshoot MTHFD2 staining in paraffin sections by checking retrieval, compartment, cell identity, and assay controls together.
Which retrieval conditions should I start with for weak MTHFD2 staining?
Start with heat-mediated retrieval in EDTA at pH 8.0 for the catalog antibody (datasheet A06465-1). This is the condition used for its paraffin-section colon cancer image, followed by antibody at 1:100 overnight at 4°C (datasheet A06465-1). If staining remains weak, compare retrieval duration on matched sections while holding antibody concentration and detection constant (standard IHC practice). Include a tissue compartment with documented expression, such as kidney tubule cells, to distinguish weak retrieval from a low-expressing sample (HPA: High in kidney tubule cells). Excessive heating can damage morphology and complicate cell-level interpretation (standard IHC practice).
Could fixation explain absent staining in my paraffin sections?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not report its fixative (datasheet A06465-1). Do not infer fixation tolerance from mitochondrial localisation or the annotated acetyllysine (UniProt P13995: mitochondrion; modified residue). Compare sections with documented processing histories using the same EDTA pH 8.0 retrieval and antibody run (datasheet A06465-1; standard IHC practice). Record fixation duration and processing differences, then inspect tissue morphology alongside signal because excessive or uneven fixation can affect antigen access in IHC generally (standard IHC practice). A documented positive control helps separate a sample-processing problem from a failed staining run (standard IHC practice).
Should diffuse cytoplasmic MTHFD2 staining count as a positive result?
MTHFD2 is mitochondrial, and its annotated sequence has no transmembrane segment (UniProt P13995: subcellular location and topology). In chromogenic sections, punctate or granular cytoplasmic staining is therefore more persuasive than isolated nuclear staining, although DAB resolution may merge closely spaced mitochondria (UniProt P13995: mitochondrion; standard IHC practice). HPA reports cytoplasmic tissue staining and supported mitochondrial localisation in cell imaging (HPA: tissue IHC profile; HPA: subcellular). Check whether the signal lies within identifiable cells and follows their cytoplasmic boundaries (standard IHC practice). If staining is uniformly diffuse, review retrieval, antibody concentration, and background controls before scoring it as specific (standard IHC practice).
How could processing or isoforms affect what this antibody detects?
MTHFD2 has 2 annotated isoforms and a processed chain spanning residues 36–350 (UniProt P13995: isoforms and processing). The supplied evidence does not map this antibody's epitope, so staining cannot establish which isoform or sequence region it recognizes (provided datasheet A06465-1; UniProt P13995: isoforms). Before comparing cases, check whether the documented immunogen or epitope overlaps sequence shared by both isoforms and the processed chain (standard antibody-validation practice). Its annotated acetyllysine at position 50 is a possible consideration only if an epitope overlaps that region; no modification-dependent staining effect is established here (UniProt P13995: modified residue).
How should I assess MTHFD2 by multiplex IF alongside this IHC result?
Use the chromogenic IHC pattern as an anatomical reference, then evaluate IF specificity independently because the supplied antibody image documents paraffin-section IHC rather than IF performance (datasheet A06465-1). Pair MTHFD2 with a validated marker of the expected cell population, such as germinal center cells in lymph node, and assess signal within that population (HPA: High in lymph node germinal center cells; standard IF practice). Select fluorophores after examining tissue autofluorescence in unstained controls (standard IF practice). Because MTHFD2 is mitochondrial and lacks a transmembrane segment, permeabilisation must provide access to an internal epitope if that is where this antibody binds; its epitope is unspecified (UniProt P13995: location and topology; provided datasheet A06465-1).
What should I change when DAB staining obscures MTHFD2-positive cells?
First inspect a section without primary antibody to assess detection-system background, and include a peroxidase-blocking step before DAB development (standard chromogenic IHC practice). The catalog image used a peroxidase-conjugated secondary and DAB, with 10% goat serum blocking and primary antibody at 1:100 (datasheet A06465-1). If background persists, titrate the primary around that documented starting concentration and review wash quality while keeping retrieval at EDTA pH 8.0 (datasheet A06465-1; standard IHC practice). Compare staining in documented positive cells with nearby cells on the same section; broad, structure-independent deposition weakens the interpretation (HPA: cell-specific tissue profile; standard IHC practice).
How should I quantify MTHFD2 staining across heterogeneous sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, then record the percentage of positive cells and staining intensity or calculate an H-score from those values (standard IHC practice). If counting discrete positive cells, report density per mm² of viable tissue rather than per whole image (standard IHC practice). Normalise comparisons to the same cell type, viable area, staining run, and exposure or scan settings (standard IHC practice). Keep glandular and immune compartments separate because HPA reports distinct immune-cell expression and high staining in several glandular populations (HPA: tissue IHC profile). Exclude folds, necrosis, and section edges using prespecified rules (standard IHC practice).
How do I distinguish true MTHFD2 signal from a staining artefact?
A plausible positive result is cell-associated cytoplasmic staining consistent with a mitochondrial protein, particularly in populations with documented expression (UniProt P13995: mitochondrion; HPA: tissue IHC profile). HPA reports High staining in lymph node germinal center cells and kidney tubule cells, while adipocytes in adipose tissue were Not detected; use these as contextual comparisons, not absolute controls for every specimen (HPA: tissue IHC). Isolated nuclear signal, edge-only deposits, and staining over necrotic material warrant review of morphology and control sections (UniProt P13995: mitochondrion; standard IHC practice). Check a no-primary control and peroxidase block when DAB deposition could reflect endogenous enzyme activity (standard chromogenic IHC practice).
Boster reagents
Best MTHFD2 / Bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase, mitochondrial IHC Antibodies
A06465-1 has IHC images from paraffin sections of human colon and lung cancers and mouse testis and epididymis; rat reactivity is listed without a tissue image (catalog: reactivity; IHC image captions).
Real IHC data
Anti-MTHFD2 Antibody
Cat # A06465-1
A06465-1 is listed for IHC and reacts with human, mouse, and rat (catalog: applications and reactivity). Its IHC images show paraffin sections of human colon cancer, human lung cancer, mouse testis, and mouse epididymis (A06465-1 IHC image captions).
Which to pick: For chromogenic IHC on paraffin sections, choose A06465-1: its captions report EDTA retrieval at pH 8.0, a 1:100 primary dilution, and peroxidase/DAB detection; the catalog separately lists an IHC dilution of 1:50 (A06465-1 IHC image captions; catalog: IHC dilution). No SKU can be recommended for IF/ICC from this payload because A06465-1 has neither an IF/ICC application listing nor an IF image (catalog: applications and IF image alts). For cross-species IHC, A06465-1 is a rabbit polyclonal listed as human, mouse, and rat reactive, though its IHC images cover human and mouse only; the fixative used for those paraffin sections is unreported (catalog: host, dilution_raw, and reactivity; A06465-1 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P13995 (MTDC_HUMAN, Bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase, mitochondrial).
- Human Protein Atlas. MTHFD2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. MTHFD2 subcellular location (ICC-IF): Localized to the mitochondria..
- Human Protein Atlas. MTHFD2 antibody validation summary (2 antibodies).
- High Expression of Methylenetetrahydrofolate Dehydrogenase 2 (MTHFD2) in Esophageal Squamous Cell Carcinoma and its Clinical Prognostic Significance. Medical science monitor : international medical journal of experimental and clinical research 2020 — PMC7055195.
- MTHFD2 marks pemetrexed resistance in pulmonary adenocarcinoma with EGFR wild type. Discover oncology 2025 — PMC12009792.
- Overexpression of MTHFD2 represents an inflamed tumor microenvironment and precisely predicts the molecular subtype and immunotherapy response of bladder cancer. Frontiers in immunology 2023 — PMC10733511.
- Down-regulation of MTHFD2 inhibits NSCLC progression by suppressing cycle-related genes. Journal of cellular and molecular medicine 2020 — PMC6991687.
- PubMed PMID:2587219 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MTHFD2 (Bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase, mitochondrial). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); mitochondrial protein (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A06465-1). Validated IHC antibody: A06465-1 at 1:50 recommended; image 1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Lymphoid tissue); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A06465-1); 2. Fixation: Image fixative and duration unreported (datasheet A06465-1); 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 A06465-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A06465-1); 8. Primary antibody: Rabbit anti-MTHFD2, 1:50 recommended; image 1:100 (datasheet A06465-1); 9. Primary incubation: Overnight at 4 °C (datasheet A06465-1). Watch for: Fixation: Keep fixation consistent across compared paraffin sections. (selected-SKU IHC image A06465-1); Caveat: Immune-cell staining can complicate mixed-tissue scoring (HPA tissue IHC); Isoform / epitope: 2 isoforms; mature chain 36–350; epitope map unspecified (UniProt); Regulation: Staining intensity varies by cell type (HPA tissue IHC). Evidence: UniProt P13995, HPA tissue IHC · Approved, HPA ICC-IF · mitochondria., Datasheet A06465-1, PMC7055195, PMC12009792. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MTHFD2/immunohistochemistry-ihc-guide.