MTHFD1L / Monofunctional C1-tetrahydrofolate synthase, mitochondrial · IHC design guide

Design Immunohistochemistry for MTHFD1L

Plan MTHFD1L staining in paraffin sections using the reported cytoplasmic tissue pattern (HPA tissue IHC) and mitochondrial annotation (UniProt). Compare high-staining colon glandular cells with low-staining prostate glandular cells, while accounting for the atlas warning that staining may reflect proteins from more than one gene (HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for MTHFD1L (IHC for MTHFD1L): expected localisation Cytoplasmic tissue staining (HPA tissue IHC); mitochondrial protein (UniProt), antibody A06289-1, validated IHC image, and IHC protocol steps — Printable **MTHFD1L** IHC protocol sheet — expected localisation Cytoplasmic tissue staining (HPA tissue IHC); mitochondrial protein (UniProt), antibody A06289-1, controls and protocol steps. Open the full MTHFD1L IHC guide →

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MTHFD1L 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	Glandular cells show cytoplasmic staining; texture unspecified (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A06289-1)
	Positive control ⓘ	Appendix+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 proteins from more than one gene (HPA tissue IHC)
	Regulation	Upregulated in colon adenocarcinoma (UniProt)
	Isoform / epitope	Two isoforms; epitope differences are unreported (UniProt)

Section 1

Recommended MTHFD1L IHC & IF Protocols

The catalog antibody uses EDTA pH 8.0 retrieval (datasheet A06289-1). The published IHC protocols below cover colorectal cancer, melanoma and bladder tissue (PMC7196253; PMC5403766; PMC6700437).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A06289-1)
Fixation	Image fixative and duration unreported (datasheet A06289-1); 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 A06289-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A06289-1)
Primary antibody	Rabbit anti-MTHFD1L, 2-5 μg/ml (datasheet A06289-1)
Primary incubation	Overnight at 4 °C (datasheet A06289-1)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A06289-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	MTHFD1L-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control.

💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A06289-1). The published protocols used pH 6 or citrate retrieval with different antibodies (PMC7196253; PMC5403766; PMC6700437).

Section 2

What Is the Expected MTHFD1L Staining Pattern?

MTHFD1L localises to mitochondria (UniProt Q6UB35; HPA ICC-IF) and should appear within the cytoplasm of expressing cells in IHC. HPA reports high staining in several glandular epithelia, kidney tubules, hepatocytes and respiratory epithelium (HPA tissue IHC). Its tissue IHC profile is Approved, with a caution that the antibody may target proteins from more than one gene; external verification is pending (HPA tissue IHC).

What am I looking at on my slide?
Cytoplasmic staining in colon or appendix glandular cells, or kidney tubular cells.	This fits HPA's reported high staining in those cells (HPA tissue IHC). A mitochondrial protein can appear as cytoplasmic signal in a chromogenic section (UniProt Q6UB35; standard IHC interpretation); IHC alone does not prove mitochondrial localisation.
Predominantly nuclear, membrane-rim or extracellular staining.	These compartments do not fit the reported mitochondrial location (UniProt Q6UB35; HPA ICC-IF). Review the counterstain and compare controls before calling the signal MTHFD1L; an unexpected compartment raises concern for artefact or nonspecific staining.
Strong staining in a cell population reported as low, while the expected cells are weak.	For example, HPA reports low staining in adipocytes and smooth muscle cells (HPA tissue IHC). This mismatch warrants a specificity check with controls or another antibody; endogenous chromogen activity can also produce misleading signal (standard IHC practice).
Similar diffuse colour across cells and surrounding tissue, with little cellular distinction.	That pattern does not match HPA's cell-specific high staining examples (HPA tissue IHC). Assess nonspecific background, detection chemistry and section quality with appropriate controls (standard IHC practice); diffuse colour alone is not evidence of MTHFD1L.
No staining in the expected cells of a known-positive section.	Check the tissue identity first: HPA reports high staining in colon glandular cells and kidney tubules (HPA tissue IHC). If controls also fail, review antibody dilution, antigen retrieval and detection steps (standard IHC practice); absence on one slide cannot establish absent protein.

💡Expected MTHFD1L appearanceA positive IHC result shows cytoplasmic signal in the expected cells, potentially strong in HPA-high glandular or tubular cells (HPA tissue IHC; UniProt Q6UB35), whereas isolated nuclear, membrane-rim or extracellular colour warrants investigation (UniProt Q6UB35 topology; HPA ICC-IF).

How each factor affects the staining
Location and topology	MTHFD1L is mitochondrial and has no annotated transmembrane segment (UniProt Q6UB35). Interpret chromogenic cytoplasmic staining with the cell type and controls; a membrane outline is not the predicted pattern.
Tissue context	HPA reports high hepatocyte IHC staining, while UniProt describes low liver expression (HPA tissue IHC; UniProt Q6UB35). Treat these as differing observations, and compare matched control sections rather than assuming either source predicts every specimen.
Antibody specificity	The tissue IHC profile is Approved but carries a more-than-one-gene targeting caution (HPA tissue IHC). Listed antibodies have Approved IHC status; none is listed as IHC Enhanced (HPA antibodies). Interpret unexpected positives conservatively.
Processing and variants	UniProt lists a mature chain spanning residues 32–978 and two isoforms (UniProt Q6UB35). These annotations alone do not predict staining intensity or establish whether a particular antibody recognises both isoforms; check the antibody's documented epitope if relevant.
IF/ICC Q: What pattern should be expected?	A: HPA reports enhanced mitochondrial localisation in ICC-IF and lists A-431, U-251MG, U2OS and MCF-7 images (HPA ICC-IF). Use that as localisation context; it does not set an IHC staining threshold or provide an IHC protocol.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Nuclear staining dominates an otherwise positive section.	The compartment conflicts with mitochondrial localisation (UniProt Q6UB35; HPA ICC-IF); the observed colour may be nonspecific or misread against the counterstain.	Compare the antibody-treated section with its detection control, then reassess the compartment at higher magnification (standard IHC practice).
Adipocytes or smooth muscle stain more strongly than nearby expected positive cells.	HPA reports low staining in these cells (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible (standard IHC practice).	Include an antibody-omission control and an independently validated antibody when available; inspect whether the same cells remain coloured (standard IHC practice).
Colour is widespread and obscures cell boundaries.	Diffuse background can arise from nonspecific binding or the chromogenic detection system (standard IHC practice); it is difficult to compare with HPA's cell-specific observations (HPA tissue IHC).	Check blocking, washes and detection controls, then score only distinguishable cellular signal (standard IHC practice).
Colon glandular cells or kidney tubules show no detectable signal.	These are reported as high in HPA tissue IHC (HPA tissue IHC); a failed staining run or specimen difference may explain the result (standard IHC practice).	Verify tissue and controls, then review the catalog antibody's IHC-P dilution, retrieval and detection instructions before repeating the run (standard IHC practice).
Hepatocyte staining seems unexpectedly strong or weak.	HPA reports high hepatocyte IHC staining, whereas UniProt reports low liver expression (HPA tissue IHC; UniProt Q6UB35); the sources do not supply a universal intensity cutoff.	Record the observed cell type and intensity, and compare sections processed in the same run (standard IHC practice).
A positive signal appears only with one antibody.	HPA cautions that its tissue IHC profile may target proteins from more than one gene, with external verification pending (HPA tissue IHC).	Review the controls and compare an independently validated antibody where available; report any disagreement rather than assigning every positive cell to MTHFD1L (standard IHC practice).

Source: UniProt Q6UB35 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing MTHFD1L staining

IHC analysis of MTHFD1L using anti-MTHFD1L antibody (A06289-1). MTHFD1L was detected in a paraffin-embedded section of human liver cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-MTHFD1L Antibody (A06289-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A06289-1 · IHCIHC analysis of MTHFD1L using anti-MTHFD1L antibody (A06289-1). MTHFD1L was detected in a paraffin-embedded section of human liver cancer ti

IF analysis of MTHFD1L using anti-MTHFD1L antibody (A06289-1). MTHFD1L was detected in an immunocytochemical section of U20S cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-MTHFD1L Antibody (A06289-1) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A06289-1 · IFIF analysis of MTHFD1L using anti-MTHFD1L antibody (A06289-1). MTHFD1L was detected in an immunocytochemical section of U20S cells. Enzyme a

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000120254-MTHFD1L/tissue/cerebral+cortex#imid_17773327

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000120254-MTHFD1L/tissue/colon#imid_17773261

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000120254-MTHFD1L/tissue/liver#imid_17773246

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000120254-MTHFD1L/tissue/kidney#imid_17773255

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA029041; green = MTHFD1L, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for MTHFD1L IHC & IF

🧪Run colon first and score its glandular cells, which have High staining (HPA: Colon glandular cells, High). HPA lists no negative tissue because MTHFD1L is detected in all 45 scored tissues (HPA); use no-primary and isotype controls for the negative comparison, and treat non-glandular cells on the colon slide as a background reference rather than confirmed target-negative cells.

Positive control tissue: Appendix (Glandular cells, HPA High)

Negative control tissue: None in HPA: MTHFD1L 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 MTHFD1L in A-431, U-251MG, U2OS, MCF-7, with annotated localisation: Mitochondria (enhanced) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only slide, a host- and clonality-matched rabbit IgG isotype control, and a MTHFD1L knockout sample as a biological negative (caption: rabbit anti-MTHFD1L antibody). Block endogenous peroxidase and check inflammatory cells for residual chromogen before scoring colon glandular staining (standard chromogenic IHC practice).

⚠️Feasibility: The selected A06289-1 paraffin-section caption does not report a fixative, and the supplied evidence reports no MTHFD1L-specific fixation window or fixation effect (selected-SKU caption). Heat retrieval in EDTA at pH 8.0 preceded staining in that caption; whether retrieval is essential is unreported (selected-SKU caption). Frozen sections are not established as easier here; ICC-IF images support a mitochondrial localisation check, while colon autofluorescence requires assessment if IF is used (HPA: mitochondria, enhanced; standard IF practice).

HPA tissue IHC evidence for MTHFD1L

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Caution, targets protein from more than one gene. 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	Glandular cells	High	Protein (IHC)	HPA →
Colon	Glandular cells	High	Protein (IHC)	HPA →
Duodenum	Glandular cells	High	Protein (IHC)	HPA →
Endometrium	Glandular cells	High	Protein (IHC)	HPA →
Kidney	Cells in tubules	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
None in HPA: MTHFD1L is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced MTHFD1L IHC Tips

Troubleshoot MTHFD1L staining in paraffin sections by checking retrieval, signal location and tissue controls before comparing expression across samples.

Which retrieval condition should I try first for MTHFD1L in paraffin sections?

Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A06289-1). The selected tissue image used that condition before staining a human liver cancer section with the catalog antibody (datasheet A06289-1). If staining is weak, adjust heating and cooling within your validated retrieval workflow while keeping EDTA and pH 8.0 as the reference condition (standard IHC practice). Compare each change against a section processed with the reference condition and include a no-primary control (standard IHC practice). Assess preserved tissue morphology alongside signal, because excessive heating can damage sections and complicate interpretation (standard IHC practice).

How should I troubleshoot weak staining when fixation history differs between blocks?

The selected paraffin-section caption does not report a fixative, so MTHFD1L-specific fixation sensitivity is unknown (datasheet A06289-1). Record each block’s fixative and fixation history before comparing its staining with another block (standard IHC practice). Process a reference section from each block with the same EDTA pH 8.0 retrieval and detection settings (datasheet A06289-1; standard IHC practice). Examine morphology and compare signal in equivalent cell populations, since tissue damage can make intensity comparisons unreliable (standard IHC practice). Do not attribute weak staining to a particular fixation effect without a controlled comparison using this antibody (standard IHC practice).

What staining pattern is plausible for MTHFD1L, and what pattern needs investigation?

MTHFD1L is assigned to mitochondria, and the subcellular imaging summary reports enhanced mitochondrial localisation (UniProt Q6UB35; HPA subcellular). In chromogenic sections, mitochondrial signal may appear as granular cytoplasmic staining when cellular detail is preserved (standard IHC practice). Compare staining within the expected cytoplasmic compartment across intact cells, rather than treating uniform nuclear colour as supportive localisation (UniProt Q6UB35; standard IHC practice). HPA describes general cytoplasmic tissue staining, which provides a broader comparison for IHC than its subcellular images (HPA tissue IHC; HPA subcellular). Recheck prominent nuclear or extracellular staining with a no-primary control and tissue morphology before interpreting it as MTHFD1L (standard IHC practice).

Can staining distinguish MTHFD1L isoforms or identify an inaccessible epitope?

The record lists two MTHFD1L isoforms, but the supplied antibody caption does not identify its epitope or establish isoform selectivity (UniProt Q6UB35; datasheet A06289-1). Therefore, report chromogenic staining as MTHFD1L-associated signal rather than assigning it to isoform 1 or 2 (UniProt Q6UB35; standard IHC practice). MTHFD1L has no annotated transmembrane segment, but that annotation does not establish which residues the antibody recognises (UniProt Q6UB35). If retrieval changes signal, compare matched sections and morphology before attributing the change to epitope exposure (standard IHC practice). Confirm isoform claims only with independent evidence that resolves the recognised sequence (standard IHC practice).

How should I assess MTHFD1L by IF alongside a cell identity marker?

Treat IF/ICC as a separate application requiring its own antibody validation; the selected evidence describes chromogenic staining in a paraffin section (datasheet A06289-1). For a colon sample, multiplex with a marker identifying glandular cells, where HPA reports high tissue staining (HPA tissue IHC). Select fluorophores after checking tissue autofluorescence and include single-channel controls to assess spectral spillover (standard IF practice). Because the antibody epitope and its side of any mitochondrial membrane are unreported, compare permeabilisation conditions and verify that they preserve cell structure while allowing antibody access (datasheet A06289-1; standard IF practice). Evaluate punctate mitochondrial colocalisation separately from diffuse background (HPA subcellular; standard IF practice).

How can I reduce diffuse DAB signal without losing plausible MTHFD1L staining?

The selected image used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and peroxidase-linked detection with DAB (datasheet A06289-1). Use those documented antibody conditions as a reference when reviewing high background in comparable paraffin sections (datasheet A06289-1; standard IHC practice). Include a no-primary section to assess secondary-reagent staining and check that endogenous peroxidase blocking is effective (standard IHC practice). Compare intact cellular signal with staining at folds, cut edges and damaged areas before changing antibody concentration (standard IHC practice). Adjust one staining variable at a time, retaining a reference section for comparison (standard IHC practice).

What is a defensible way to score MTHFD1L chromogenic staining? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and tissue area before scoring, and exclude folds, necrosis and poorly preserved regions (standard IHC practice). For intact cells, record the percentage in each intensity category and calculate an H-score as 1 × weak% + 2 × moderate% + 3 × strong% (standard IHC practice). Alternatively, report the percentage of positive cells or positive-cell density per mm², with the threshold and denominator stated (standard IHC practice). Normalise to eligible cells or viable tissue area, and use the same retrieval, detection and scoring rules across samples (standard IHC practice). HPA’s high hepatocyte staining and UniProt’s low liver-expression description come from different evidence summaries, so document the reference used for comparisons (HPA tissue IHC; UniProt Q6UB35).

When should a positive MTHFD1L IHC result be treated cautiously?

Give greatest weight to staining in intact cells with a plausible cytoplasmic pattern, consistent with the mitochondrial assignment and HPA tissue profile (UniProt Q6UB35; HPA tissue IHC). Investigate dominant nuclear, extracellular or unexpected cell-population staining before calling it MTHFD1L (UniProt Q6UB35; standard IHC practice). Edge staining, necrotic areas and residual endogenous peroxidase activity can produce misleading DAB signal; assess morphology and the no-primary control (standard IHC practice). HPA reports high staining in several epithelial populations but cautions that its tissue antibody targets protein from more than one gene and awaits external verification (HPA tissue IHC). Interpret strong staining with that specificity limitation in mind and seek independent confirmation for consequential conclusions (HPA tissue IHC; standard IHC practice).

Boster reagents

Best MTHFD1L / Monofunctional C1-tetrahydrofolate synthase, mitochondrial IHC Antibodies

A06289-1 has IHC data from a paraffin-embedded human liver cancer section and IF data from U20S cells (catalog image captions). Its listed reactivity covers human, mouse and rat (catalog: reactivity).

Real IHC data

Anti-MTHFD1L Antibody ®

Cat # A06289-1

View product →

A06289-1 is listed for IHC and IF, with human, mouse and rat reactivity (catalog: applications and reactivity). Its images show IHC in a paraffin-embedded human liver cancer section and IF in U20S cells (catalog image captions).

Which to pick: Choose A06289-1 for paraffin-section IHC: its IHC image used heat retrieval in EDTA at pH 8.0 and 2 μg/ml primary antibody (A06289-1 IHC image caption). Choose the same SKU for IF/ICC because both applications are listed and its IF image shows staining in U20S cells (catalog: applications; A06289-1 IF image caption). For mouse or rat work, A06289-1 lists reactivity with both species, while the supplied IHC image shows human tissue; the IHC caption does not report the fixative (catalog: reactivity; A06289-1 IHC image caption).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry Q6UB35 (C1TM_HUMAN, Monofunctional C1-tetrahydrofolate synthase, mitochondrial).
Human Protein Atlas. MTHFD1L tissue IHC expression (reliability: Approved).
Human Protein Atlas. MTHFD1L subcellular location (ICC-IF): Localized to the mitochondria..
Human Protein Atlas. MTHFD1L antibody validation summary (5 antibodies).
High expression of folate cycle enzyme MTHFD1L correlates with poor prognosis and increased proliferation and migration in colorectal cancer. Journal of Cancer 2020 — PMC7196253.
Affinity Proteomics Exploration of Melanoma Identifies Proteins in Serum with Associations to T-Stage and Recurrence. Translational oncology 2017 — PMC5403766.
Expression and Role of Methylenetetrahydrofolate Dehydrogenase 1 Like (MTHFD1L) in Bladder Cancer. Translational oncology 2019 — PMC6700437.
MTHFD1L in muscle invasive bladder cancer: a multi-cohort study on prognosis and therapeutic response. World journal of surgical oncology 2026 — PMC13192160.
PubMed PMID:12937168 — UniProt-cited evidence.
PubMed PMID:15013446 — UniProt-cited evidence.
PubMed PMID:14574404 — UniProt-cited evidence.