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MTHFD1 / C-1-tetrahydrofolate synthase, cytoplasmic · IHC design guide
Design Immunohistochemistry for MTHFD1
Plan chromogenic IHC for MTHFD1 in paraffin sections using the catalog antibody at 2–5 μg/mL (datasheet A02025-1). Expect cytoplasmic staining, strongest in hepatocytes, while interpreting specificity with care (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MTHFD1 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 staining in tissue (HPA tissue IHC) | |
| Staining pattern | Hepatocytes show strong cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A02025-1) | |
| Positive control | Liver+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) | |
| Caveat | Staining may reflect more than one gene (HPA tissue IHC) | |
| Regulation | Liver-enriched RNA expression (HPA tissue RNA) | |
| Isoform / epitope | No isoforms; loss of residue 1 may affect N-terminal epitopes (UniProt) |
Section 1
Recommended MTHFD1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A02025-1) is followed by published MTHFD1 IHC methods from four articles (PMC11288495; PMC7947712; PMC11581708; PMC8264328).
| Sample | Paraffin-embedded human adenocarcinoma of the lung tissue; fixative not specified (datasheet A02025-1) |
| Fixation | Image fixative and duration unreported (datasheet A02025-1); 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 A02025-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A02025-1) |
| Primary antibody | Rabbit anti-MTHFD1, 2-5 μg/ml (datasheet A02025-1) |
| Primary incubation | Overnight at 4 °C (datasheet A02025-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A02025-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | MTHFD1-positive staining in hepatocytes of liver (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression, most abundant in liver. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: A02025-1); optimize retrieval for other antibodies (PMC7947712; PMC8264328).
Section 2
What Is the Expected MTHFD1 Staining Pattern?
MTHFD1 should show cytoplasmic staining across many tissues, with the strongest listed IHC signal in hepatocytes (UniProt P11586: cytoplasm and no transmembrane segment; HPA: general cytoplasmic expression, high in hepatocytes). HPA rates its tissue IHC evidence Approved but reports low agreement with RNA data and cautions that staining may include proteins from more than one gene (HPA: tissue IHC reliability).
| Hepatocytes show strong cytoplasmic chromogen; nearby tissue is less intense. | This fits the strongest listed tissue pattern: high hepatocyte staining and general cytoplasmic expression (HPA: tissue IHC). Compare cell compartments and tissue context before assigning a score. |
| A sharply nuclear, membranous or extracellular signal dominates the section. | The dominant compartment conflicts with cytoplasmic MTHFD1 localization (UniProt P11586: cytoplasm; HPA: tissue IHC). Consider an artifact or another detected protein; compartment alone cannot establish antibody specificity (HPA: tissue IHC reliability). |
| A low-staining cell group appears stronger than hepatocytes in the same run. | Check whether the signal follows tissue structures or the detection system. HPA lists cardiomyocytes and skeletal myocytes as low, versus high hepatocytes (HPA: tissue IHC). Cross-reactivity or endogenous chromogen-forming activity is possible (HPA: tissue IHC reliability; general IHC practice). |
| Chromogen forms a diffuse haze across cells, stroma and blank areas. | A haze without cell-shaped cytoplasmic contrast is difficult to score as MTHFD1 (HPA: general cytoplasmic expression; general IHC practice). Nonspecific antibody binding or detection background may contribute (general IHC practice). |
| A liver section shows no discernible hepatocyte signal. | This conflicts with the reported high hepatocyte staining (HPA: tissue IHC). Review run controls and tissue integrity before calling the sample negative (general IHC practice); HPA's Approved rating does not guarantee every antibody or run will reproduce its pattern (HPA: tissue IHC reliability). |
💡Expected MTHFD1 appearanceCall a positive result when hepatocytes show strong, cell-shaped cytoplasmic staining (HPA: high in hepatocytes; UniProt P11586: cytoplasm); dominant nuclear, membranous or structure-free haze is suspect (UniProt P11586: cytoplasm; general IHC practice).
| Tissue and cell selection | Liver hepatocytes provide the strongest listed reference; HPA reports medium staining in several other listed cell types and low staining in selected muscle cells (HPA: tissue IHC). Low is not an established negative control. |
| Antibody interpretation | Four listed antibodies have Approved IHC status, while HPA reports low staining–RNA agreement and possible detection of protein from more than one gene (HPA: antibody list; tissue IHC reliability). Treat staining intensity as evidence to check, not proof of identity. |
| Compartment and protein form | MTHFD1 is cytoplasmic, lacks a transmembrane segment and has recorded 1–935 and N-terminally processed 2–935 chains (UniProt P11586). The supplied record gives no antibody epitope, so the processing entry cannot predict an antibody-specific staining difference. |
| Chromogenic detection | Endogenous enzyme activity or nonspecific reagent binding can add color independently of target staining (general IHC practice). Interpret chromogen against a suitable detection control, particularly when color lacks cytoplasmic cell boundaries (general IHC practice). |
| IF/ICC Q: Where should MTHFD1 appear? | A: In the cytosol (HPA: subcellular ICC-IF, enhanced). That observation supports compartment interpretation here; IF/ICC setup belongs to its separate guide and does not establish a chromogenic IHC intensity threshold. |
| Situation | Likely cause | Next action |
|---|---|---|
| No hepatocyte staining in the liver reference section. | The run may have failed, tissue quality may be poor, or the antibody may not perform under the chosen conditions (general IHC practice). | Check the IHC-validated antibody's instructions, a known-positive section and detection controls; revisit antigen retrieval only as a general IHC optimization step, without assuming a documented MTHFD1 fixation effect (HPA: high in hepatocytes; general IHC practice). |
| Strong signal appears mainly in nuclei or along membranes. | That dominant pattern disagrees with the reported cytoplasmic location; off-target staining or an interpretation artifact is possible (UniProt P11586: cytoplasm; HPA: tissue IHC reliability). | Compare compartment boundaries with the counterstain and an appropriate control, then confirm the pattern with an independent antibody if available (general IHC practice; HPA: multiple listed IHC antibodies). |
| Low-listed muscle cells appear intensely positive. | The result departs from HPA's low staining in cardiomyocytes and skeletal myocytes; cross-reactivity or detection background may contribute (HPA: tissue IHC and reliability; general IHC practice). | Compare the cells with a liver reference and detection control in the same run; investigate reagent background before assigning a biological difference (HPA: high in hepatocytes; general IHC practice). |
| Diffuse chromogen obscures cell boundaries. | Nonspecific binding, incomplete blocking or endogenous detection activity can create broad background (general IHC practice). | Inspect the detection-only control, review blocking and antibody dilution, and score only interpretable cell-shaped cytoplasmic signal (general IHC practice; HPA: general cytoplasmic expression). |
| Staining is weak across every section. | A shared retrieval, antibody incubation or detection problem is possible (general IHC practice); HPA does not establish MTHFD1-specific fixation sensitivity (HPA: tissue IHC scope). | Compare a known-positive liver section and run controls, then adjust one general IHC step at a time according to the antibody instructions (HPA: high in hepatocytes; general IHC practice). |
| Two antibodies yield different cell patterns. | HPA reports low agreement with RNA data and cautions about detection of protein from more than one gene, despite Approved IHC ratings (HPA: tissue IHC reliability; antibody list). | Compare their cytoplasmic patterns in liver and the same tissue panel; document the discrepancy and avoid calling an antibody-specific pattern definitive without further validation (HPA: high in hepatocytes; general IHC practice). |
Experiment evidence
IHC & IF images showing MTHFD1 staining
A02025-1 · IHCIHC analysis of MTHFD1 using anti-MTHFD1 antibody (A02025-1). MTHFD1 was detected in a paraffin-embedded section of human adenocarcinoma of
A02025-1 · IHCIHC analysis of MTHFD1 using anti-MTHFD1 antibody (A02025-1). MTHFD1 was detected in a paraffin-embedded section of human chronic tonsilliti
A02025-1 · IFIF analysis of MTHFD1 using anti-MTHFD1 antibody (A02025-1). MTHFD1 was detected in an immunocytochemical section of Hela cells. Enzyme anti
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000100714-MTHFD1/tissue/cerebral+cortex#imid_17773327
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000100714-MTHFD1/tissue/colon#imid_17773261
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000100714-MTHFD1/tissue/liver#imid_17773246
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000100714-MTHFD1/tissue/kidney#imid_17773247
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001290; green = MTHFD1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001290; green = MTHFD1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MTHFD1 IHC & IF
🧪Run liver first: hepatocytes should show MTHFD1 staining (HPA: High in hepatocytes). HPA detects MTHFD1 in all 45 scored tissues, so there is no supported negative tissue or established negative cell population within the liver slide; use no-primary and isotype controls to assess background, including staining in noncellular spaces (HPA: detected in all 45 scored tissues).
Positive control tissue: Liver (Hepatocytes, HPA High)
Negative control tissue: None in HPA: MTHFD1 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 MTHFD1 in A-431, U-251MG, U2OS, KOLF2.1J, NIH 3T3, with annotated localisation: Cytosol (enhanced) (HPA subcellular).
Technical controls: Run a secondary-only slide and a rabbit isotype control matched to the catalog antibody, then confirm specificity with MTHFD1 knockout material or a peptide-block control if the immunizing peptide is available (caption: rabbit anti-MTHFD1 antibody; standard IHC practice). For liver DAB staining, quench endogenous peroxidase and inspect the control slides for pigment or other background (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A02025-1 paraffin-section caption does not state the fixative (selected-SKU tissue-IHC caption). The caption reports heat retrieval in EDTA at pH 8.0, but one reported condition does not establish whether retrieval is required (selected-SKU tissue-IHC caption). HPA shows cytosolic ICC-IF localization, but the supplied evidence does not establish whether IF or frozen sections are easier for MTHFD1; liver pigment and endogenous peroxidase can complicate chromogenic scoring (HPA: cytosol, enhanced; standard IHC practice).
HPA tissue IHC evidence for MTHFD1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene.). 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 |
|---|---|---|---|---|
| Liver | Hepatocytes | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: MTHFD1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced MTHFD1 IHC Tips
Troubleshoot cytoplasmic MTHFD1 staining in paraffin section IHC using the catalog antibody’s documented conditions and compartment specific controls.
What should I change if MTHFD1 staining is weak after antigen retrieval?
Start with heat mediated retrieval in EDTA, pH 8.0 (datasheet A02025-1). The documented paraffin section workflow then used 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C (datasheet A02025-1). Check that retrieval heating and cooling were consistent across sections before changing primary antibody concentration (standard IHC practice). If staining remains weak, compare a small retrieval time series on matched sections while keeping detection and exposure conditions fixed (standard IHC practice). Include liver hepatocytes as a tissue reference, while recognizing that HPA reports low consistency between staining and RNA data (HPA: High in hepatocytes; HPA: reliability description).
Could fixation explain weak or uneven MTHFD1 staining?
The catalog image describes a paraffin section but does not state its fixative, so target specific fixation sensitivity is unknown (datasheet A02025-1). Record the fixative, fixation duration, tissue thickness and processing history for every section before comparing staining intensity (standard IHC practice). Assess matched sections processed together, using the documented EDTA, pH 8.0 retrieval and 2 μg/ml primary concentration as reference conditions (datasheet A02025-1). If staining varies across a specimen, inspect morphology and compare central and peripheral areas before assigning the difference to MTHFD1 expression (standard IHC practice). HPA tissue patterns cannot establish a fixation effect for this antibody (HPA: tissue IHC profile).
Where should convincing MTHFD1 staining appear in a tissue section?
Expect predominantly cytoplasmic staining: MTHFD1 is annotated in the cytoplasm, and cellular imaging places it in the cytosol (UniProt P11586: subcellular location; HPA: cytosol). Its record lists no transmembrane segment, so a sharply membrane restricted pattern warrants investigation rather than automatic acceptance (UniProt P11586: topology). Compare stained cells with adjacent unstained structures on the same section and use a nuclear counterstain to distinguish cytoplasm from nuclei (standard IHC practice). Liver hepatocytes provide a useful positive tissue reference, although their staining intensity is not a universal cutoff (HPA: High in hepatocytes; HPA: reliability description). Verify unexpected nuclear or membrane patterns with an independent specificity control (standard IHC practice).
Can isoforms or modifications change what this antibody detects in IHC?
No alternative isoforms are annotated for MTHFD1, while the record lists full length and N terminally processed chains (UniProt P11586: isoforms and processing). Reported modifications include N terminal acetylation and phosphorylation at residues 318, 413 and 490 (UniProt P11586: modified residues). These annotations do not identify this antibody’s binding site or show that a modification alters its staining (UniProt P11586: modified residues; datasheet A02025-1: epitope not stated). If staining changes between specimens, first compare matched retrieval, detection and tissue preservation conditions (standard IHC practice). Seek antibody epitope information or use an independently validated antibody before assigning a pattern to processing or phosphorylation (standard IHC practice).
How can I check a cytoplasmic MTHFD1 pattern by multiplex IF?
For a liver comparison, pair MTHFD1 with an independently validated hepatocyte marker and assess cytosolic signal within the marker defined cells (HPA: High in hepatocytes; HPA: cytosol). Choose a fluorophore in a channel with low tissue autofluorescence, and inspect unstained tissue in every planned channel before setting exposure (standard IF practice). Because MTHFD1 is cytosolic and has no transmembrane segment, an intracellular staining workflow needs permeabilisation that permits antibody access to the cytosol (HPA: cytosol; UniProt P11586: topology; standard IF practice). Titrate permeabilisation against preservation of cell boundaries and include single stain controls when assessing overlap (standard IF practice). The paraffin section caption supplies no IF fixation or permeabilisation condition (datasheet A02025-1).
How do I separate diffuse MTHFD1 staining from chromogenic background?
MTHFD1 can produce a broad cytoplasmic pattern, so diffuse color alone does not establish specific staining (HPA: general cytoplasmic expression; standard IHC practice). Compare a primary omitted control with the test section to identify signal from detection reagents and endogenous peroxidase (standard IHC practice). Apply a peroxidase block before HRP and develop DAB for the same controlled interval across compared sections (standard chromogenic IHC practice). The documented workflow used 10% goat serum, 2 μg/ml primary overnight at 4°C, and an HRP based DAB readout (datasheet A02025-1). If background persists, assess blocking, washing and primary concentration on matched sections while preserving the positive reference (standard IHC practice).
How should I quantify MTHFD1 across differently cellular tissue regions? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and cytoplasmic compartment before measuring MTHFD1 signal (HPA: cytosol; standard IHC practice). For cell based comparisons, report the percentage of positive cells and an intensity weighted H-score, using one prespecified threshold across slides (standard IHC scoring practice). If cell density differs between regions, report positive cells per mm² of viable tissue or normalize positive cells to the counted cells in the same region (standard IHC scoring practice). Exclude folds, necrosis and section edges using documented criteria before scoring (standard IHC practice). Keep retrieval, DAB development and image acquisition consistent, and interpret intensity cautiously because HPA reports low agreement between staining and RNA expression (standard IHC practice; HPA: reliability description).
Which staining patterns support a true MTHFD1 positive result?
A credible result shows reproducible cytoplasmic signal in intact cells, consistent with MTHFD1’s cytosolic annotation (UniProt P11586: cytoplasm; HPA: cytosol). Liver hepatocytes are a reference population with reported high staining, although HPA cautions that antibody staining and RNA expression have low consistency (HPA: High in hepatocytes; HPA: reliability description). Treat staining confined to nuclei, membranes, damaged tissue or section edges as a prompt to inspect morphology and controls (UniProt P11586: topology; standard IHC practice). Distinguish cellular signal from endogenous peroxidase with a primary omitted control and an appropriate peroxidase block (standard chromogenic IHC practice). HPA also cautions that its tissue staining may recognize proteins from more than one gene, so confirm consequential assignments independently (HPA: reliability description).
Boster reagents
Best MTHFD1 / C-1-tetrahydrofolate synthase, cytoplasmic IHC Antibodies
A02025-1 has human paraffin-section IHC images and HeLa-cell and human tonsil IF images (catalog image captions). Its listed reactivity covers human, mouse, and rat (catalog reactivity).
Real IHC data
Anti-MTHFD1 Antibody ®
Cat # A02025-1
A02025-1 has IHC images from paraffin sections of human lung adenocarcinoma, chronic tonsillitis, diffuse large B-cell lymphoma, and liver cancer (catalog IHC captions). It also has IF images from HeLa cells and a paraffin section of human tonsil (catalog IF captions).
Which to pick: Choose A02025-1 for tissue IHC: its human paraffin-section captions document EDTA retrieval at pH 8.0 and primary antibody at 2 μg/ml (catalog IHC captions); the fixative is unreported (catalog IHC captions). The same SKU supports IF/ICC, with images from HeLa cells and human tonsil using 5 μg/ml primary antibody (catalog applications; catalog IF captions). For cross-species planning, A02025-1 lists human, mouse, and rat reactivity, while its supplied IHC and IF images show human samples only (catalog reactivity; catalog image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P11586 (C1TC_HUMAN, C-1-tetrahydrofolate synthase, cytoplasmic).
- Human Protein Atlas. MTHFD1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. MTHFD1 subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. MTHFD1 antibody validation summary (4 antibodies).
- Specific association of MTHFD1 expressions with small cell lung cancer development and chemoradiotherapy outcome. Saudi medical journal 2024 — PMC11288495.
- Mining of RNA Methylation-Related Genes and Elucidation of Their Molecular Biology in Gallbladder Carcinoma. Frontiers in oncology 2021 — PMC7947712.
- MTHFD1 Regulates Autophagy to Promote Growth and Metastasis in Colorectal Cancer via the PI3K-AKT-mTOR Signaling Pathway. Cancer medicine 2024 — PMC11581708.
- One carbon metabolism in human lung cancer. Translational lung cancer research 2021 — PMC8264328.
- PubMed PMID:3053686 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:12508121 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MTHFD1 (C-1-tetrahydrofolate synthase, cytoplasmic). Expected localisation Cytoplasmic staining in tissue (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A02025-1). Validated IHC antibody: A02025-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Liver (Hepatocytes); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human adenocarcinoma of the lung tissue; fixative not specified (datasheet A02025-1); 2. Fixation: Image fixative and duration unreported (datasheet A02025-1); 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 A02025-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A02025-1); 8. Primary antibody: Rabbit anti-MTHFD1, 2-5 μg/ml (datasheet A02025-1); 9. Primary incubation: Overnight at 4 °C (datasheet A02025-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining may reflect more than one gene (HPA tissue IHC); Isoform / epitope: No isoforms; loss of residue 1 may affect N-terminal epitopes (UniProt); Regulation: Liver-enriched RNA expression (HPA tissue RNA). Evidence: UniProt P11586, HPA tissue IHC · Approved, HPA ICC-IF · cytosol., Datasheet A02025-1, PMC11288495, PMC7947712. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MTHFD1/immunohistochemistry-ihc-guide.