MCU / Calcium uniporter protein, mitochondrial · IHC design guide

Design Immunohistochemistry for MCU

Plan chromogenic paraffin-section MCU staining using the observed cytoplasmic tissue pattern (HPA tissue IHC) and its mitochondrial inner membrane location (UniProt). Start the IHC-validated antibody at 2–5 μg/ml (datasheet A00685-1), and compare cellular staining with matched controls (standard IHC practice).

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

Immunohistochemistry protocol sheet for MCU (IHC for MCU): expected localisation Cytoplasm (HPA tissue IHC); mitochondrial inner membrane (UniProt), antibody A00685-1, validated IHC image, and IHC protocol steps — Printable **MCU** IHC protocol sheet — expected localisation Cytoplasm (HPA tissue IHC); mitochondrial inner membrane (UniProt), antibody A00685-1, controls and protocol steps. Open the full MCU IHC guide →

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MCU 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	Cytoplasm (HPA tissue IHC); mitochondrial inner membrane (UniProt)
	Staining pattern	Cytoplasmic staining in most tissues (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A00685-1)
	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)
	Caveat	Staining has low agreement with RNA expression (HPA tissue IHC)
	Regulation	Expression regulation is not specified (UniProt)
	Isoform / epitope	3 isoforms; mature chain 51–351; epitope map unspecified (UniProt)

Section 1

Recommended MCU IHC & IF Protocols

The catalog antibody’s IHC-P protocol (datasheet A00685-1) is accompanied by three published MCU tissue IHC protocols (PMC7680189; PMC8805845; PMC8195654).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded mouse lung tissue; fixative not specified (datasheet A00685-1)
Fixation	Image fixative and duration unreported (datasheet A00685-1); 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 A00685-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A00685-1)
Primary antibody	Rabbit anti-MCU, 2-5μg/ml (datasheet A00685-1)
Primary incubation	Overnight at 4 °C (datasheet A00685-1)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A00685-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	MCU-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet A00685-1). Citric-acid microwave retrieval is a published alternative (PMC8805845).

Section 2

What Is the Expected MCU Staining Pattern?

MCU resides in the mitochondrial inner membrane, with matrix-facing regions on either side of two transmembrane segments (UniProt Q8NE86 topology). In paraffin-section IHC, expect cytoplasmic staining in many cell types, including glandular and neuronal cells (HPA tissue IHC). HPA rates tissue IHC Approved but reports low consistency between antibody staining and RNA expression, so interpret intensity with that limitation (HPA tissue IHC).

What am I looking at on my slide?
Cytoplasmic staining in adrenal or colon glandular cells, bronchial basal cells, or cortical neurons.	These cell types have High staining in HPA tissue IHC (HPA tissue IHC). A cytoplasmic pattern fits MCU's mitochondrial location (UniProt Q8NE86; HPA tissue IHC). Chromogenic IHC cannot establish staining of the inner membrane itself from the slide pattern alone (general IHC interpretation).
Strong, predominantly nuclear or cell-surface staining, with little cytoplasmic signal.	That compartment does not fit MCU's mitochondrial inner membrane location (UniProt Q8NE86) or HPA's cytoplasmic tissue profile (HPA tissue IHC). Treat it as a possible staining artefact and reassess antibody specificity and detection controls before scoring it as MCU (general IHC practice).
Strong staining in oral-mucosa squamous cells or soft-tissue fibroblasts.	HPA reports Low staining in these cells (HPA tissue IHC). An unexpectedly strong result warrants checks for cross-reactivity or endogenous detection activity (general IHC practice). Low is not absent: the HPA record supplies no negative tissue or cell population for a strict absence criterion (HPA tissue IHC).
Broad, even colour across tissue, including areas without clear cellular boundaries.	This does not resemble HPA's cell-associated cytoplasmic profile (HPA tissue IHC). Background from detection reagents, insufficient blocking, or excess antibody can obscure the cellular pattern (general IHC practice); do not score diffuse colour as a positive cell signal.
No detectable staining in adrenal glandular cells or colon glandular cells.	Both are reported High in HPA tissue IHC (HPA tissue IHC), so an absent result calls for a review of staining and detection controls (general IHC practice). HPA's Approved rating carries a low staining-versus-RNA consistency caveat; one failed section does not establish true absence of MCU (HPA tissue IHC).

💡Expected MCU appearanceCall a result positive when cell-associated cytoplasmic colour is clear in cells reported High, such as adrenal or colon glandular cells (HPA tissue IHC), and treat dominant nuclear, cell-surface, or diffuse tissue-wide colour as suspect because MCU is mitochondrial (UniProt Q8NE86; general IHC interpretation).

How each factor affects the staining
Topology and epitope location	MCU has two transmembrane segments at residues 234–255 and 263–284, with matrix and intermembrane regions (UniProt Q8NE86 topology). Epitope location may affect antibody access, but no epitope or target-specific retrieval response is supplied.
Protein processing	UniProt annotates a 51–351 chain and no propeptide (UniProt Q8NE86 processing). The record does not support a shed extracellular staining pattern; use the mitochondrial location to assess compartment fit (UniProt Q8NE86 subcellular location).
Isoforms	Three isoforms are annotated (UniProt Q8NE86). Their presence alone does not establish which forms the IHC antibody detects or whether tissue staining should differ between isoforms; that requires antibody epitope or validation evidence.
Evidence strength	HPA lists antibody HPA016480 as IHC Approved, while the tissue profile has low consistency with RNA expression (HPA antibody validation; HPA tissue IHC). Use the listed cell patterns as reference observations, with cautious interpretation of disagreements.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A reported High cell population shows no stain.	Staining or detection may have failed; HPA reports High glandular-cell staining in adrenal gland and colon (HPA tissue IHC; general IHC practice).	Check a concurrent positive tissue, reagent sequence, antibody dilution, retrieval conditions, and chromogen development; change conditions only with appropriate controls (general IHC practice).
Colour is diffuse across cells and extracellular areas.	Non-specific reagent binding or excess chromogen development can create background (general IHC practice).	Review blocking, washes, antibody dilution, and development time; compare with a detection-only control before assigning cellular staining (general IHC practice).
Nuclei or cell surfaces dominate the signal.	The pattern conflicts with mitochondrial inner membrane localisation (UniProt Q8NE86) and cytoplasmic tissue staining (HPA tissue IHC).	Review section morphology and detection controls; seek concordant cytoplasmic staining before calling MCU positive (general IHC practice).
Low-reference cells stain as strongly as High-reference cells.	Oral-mucosa squamous cells and soft-tissue fibroblasts are Low, while colon glandular cells are High (HPA tissue IHC); broad signal may reflect background (general IHC practice).	Compare matched sections under the same development conditions and inspect detection-only controls; avoid treating Low as a negative control (HPA tissue IHC; general IHC practice).
IHC intensity disagrees with RNA expectations.	HPA explicitly reports low consistency between antibody staining and RNA expression data despite its Approved IHC rating (HPA tissue IHC).	Record the cell type, compartment, and control performance; interpret the disagreement cautiously instead of converting RNA abundance into an expected IHC score (HPA tissue IHC; general IHC interpretation).
Q: Can IF/ICC help assess localisation?	HPA reports supported mitochondrial localisation by ICC-IF and lists HPA016480 as ICC Supported (HPA subcellular; HPA antibody validation).	A: Compare localisation evidence in the separate IF/ICC guide; use it to assess compartment plausibility, while scoring paraffin-section IHC against its own tissue observations and controls (HPA subcellular; HPA tissue IHC; general IHC practice).

Source: UniProt Q8NE86 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing MCU staining

IHC analysis of MCU using anti-MCU antibody (A00685-1). MCU was detected in paraffin-embedded section of mouse lung tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-MCU Antibody (A00685-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

A00685-1 · IHCIHC analysis of MCU using anti-MCU antibody (A00685-1). MCU was detected in paraffin-embedded section of mouse lung tissue. Heat mediated an

IHC analysis of MCU using anti-MCU antibody (A00685-1). MCU was detected in paraffin-embedded section of rat lung tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-MCU Antibody (A00685-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

A00685-1 · IHCIHC analysis of MCU using anti-MCU antibody (A00685-1). MCU was detected in paraffin-embedded section of rat lung tissue. Heat mediated anti

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000156026-MCU/tissue/cerebral+cortex#imid_4352072

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000156026-MCU/tissue/colon#imid_4351990

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000156026-MCU/tissue/liver#imid_4352007

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000156026-MCU/tissue/kidney#imid_4352000

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA016480; green = MCU, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for MCU IHC & IF

🧪Start with adrenal gland: its glandular cells should show MCU staining (HPA: High in adrenal gland glandular cells). HPA detects MCU in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and treat weakly stained neighboring cells as contrast rather than confirmed MCU-negative cells (HPA: no negative rows; detected in all 45 scored tissues).

Positive control tissue: Adrenal gland (Glandular cells, HPA High)

Negative control tissue: None in HPA: MCU 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 MCU in A-431, U-251MG, U2OS, with annotated localisation: Mitochondria (supported) (HPA subcellular).

Technical controls: Run a secondary-only control and a concentration-matched rabbit IgG control matched to the primary antibody’s clonality, plus MCU knockout tissue or a validated immunizing-peptide block if available (caption: rabbit anti-MCU antibody; standard IHC control practice). Quench endogenous peroxidase and check adrenal pigment against the brown DAB signal (caption: DAB detection; standard IHC practice).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A00685-1 paraffin-section caption does not state the fixative (caption: fixative unreported). That caption uses heat retrieval in EDTA at pH 8.0, but does not establish retrieval dependence or show that frozen sections or IF are easier (caption: EDTA retrieval; no comparison reported). Adrenal pigment may complicate DAB interpretation; mitochondrial localisation provides a useful staining check (standard IHC practice; UniProt Q8NE86: mitochondrial inner membrane).

HPA tissue IHC evidence for MCU

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 →
Breast	Glandular cells	High	Protein (IHC)	HPA →
Bronchus	Basal 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: MCU is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced MCU IHC Tips

Troubleshoot chromogenic MCU staining in paraffin sections by checking retrieval, cellular pattern, controls, and scoring before interpreting biological differences.

What retrieval should I start with for MCU in paraffin sections?

Start with heat-mediated retrieval in EDTA at pH 8.0 for this antibody (datasheet A00685-1). The selected mouse lung section was stained after that retrieval, followed by 10% goat serum blocking and 2 µg/ml primary antibody overnight at 4°C (IHC caption A00685-1). If staining is weak, first compare a fresh section with the documented workflow and include a known staining control on the same run (standard IHC practice). Vary heating duration cautiously, because excessive retrieval can damage section morphology and make mitochondrial staining difficult to judge (standard IHC practice).

Could fixation explain weak or uneven MCU staining?

Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (IHC caption A00685-1). Record fixation method and duration for each specimen, then compare sections processed together before attributing different staining intensities to MCU abundance (standard IHC practice). Check whether weak areas coincide with poor tissue preservation or section loss after heat retrieval in EDTA at pH 8.0 (datasheet A00685-1; standard IHC practice). Keep retrieval, 2 µg/ml primary concentration, and overnight incubation at 4°C consistent while assessing fixation as a possible source of variation (IHC caption A00685-1; standard IHC practice).

Where should convincing MCU signal appear in chromogenic tissue sections?

MCU is assigned to the mitochondrial inner membrane, with 2 transmembrane segments at residues 234–255 and 263–284 (UniProt Q8NE86 topology). In chromogenic sections, assess a cytoplasmic, potentially granular pattern in intact cells; light microscopy generally cannot resolve the inner membrane itself (HPA tissue IHC profile; standard IHC practice). Compare the pattern with a mitochondrial marker on an adjacent section when compartment assignment matters (UniProt Q8NE86 subcellular location; standard IHC practice). Strong nuclear-only or extracellular DAB signal does not fit the assigned location and merits review of controls, tissue preservation, and chromogen deposits (UniProt Q8NE86 subcellular location; standard IHC practice).

How do MCU isoforms and membrane topology affect epitope interpretation?

The record lists 3 MCU isoforms, but the supplied antibody evidence does not identify which isoforms its epitope recognizes (UniProt Q8NE86 isoforms; IHC caption A00685-1). Ask for the immunogen or mapped epitope before describing staining as isoform-specific (standard IHC practice). The mature chain spans residues 51–351, with matrix-facing regions at 51–233 and 285–351 and an intermembrane-space segment at 256–262 (UniProt Q8NE86 processing and topology). If sections stain inconsistently, test retrieval and antibody specificity with appropriate controls; topology alone cannot establish which epitope remains accessible after processing (UniProt Q8NE86 topology; standard IHC practice).

How should I use IF to investigate an ambiguous MCU IHC pattern?

Use IF as a separate localisation check: MCU is assigned to mitochondria, and supported mitochondrial localisation is reported in cell imaging (UniProt Q8NE86 subcellular location; HPA subcellular). Multiplex MCU with a mitochondrial marker and a marker for the cell population being evaluated, assigning spectrally distinct fluorophores and checking unstained tissue for autofluorescence (standard IF practice). Choose permeabilisation after determining the antibody epitope: MCU has matrix-facing regions at 51–233 and 285–351 and an intermembrane-space segment at 256–262 (UniProt Q8NE86 topology; standard IF practice). Keep single-label and secondary-only controls so bleed-through or nonspecific fluorescence cannot masquerade as colocalisation (standard IF practice).

What should I check when MCU DAB staining is diffuse or excessive?

Start with a no-primary control to identify signal from detection reagents, and inspect areas with heavy pigment or poor preservation separately (standard IHC practice). The documented workflow uses a biotinylated secondary, streptavidin-biotin complex, and DAB, so evaluate endogenous biotin and peroxidase contributions with appropriate blocking controls (IHC caption A00685-1; standard IHC practice). Compare background after the documented 10% goat serum block and 2 µg/ml primary incubation overnight at 4°C (IHC caption A00685-1). If background persists, adjust blocking, washing, or antibody concentration systematically while checking that expected cytoplasmic staining remains distinguishable (HPA tissue IHC profile; standard IHC practice).

How should I score MCU staining across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and region before scoring, and exclude folds, necrosis, and section edges by the same rule for every specimen (standard IHC practice). Record the percentage of positive cells and staining intensity, or calculate an H-score from intensity categories 0–3 and their cell percentages (standard IHC practice). For spatial comparisons, report positive-cell density per mm² of viable tissue and normalise to the number of eligible cells or viable tissue area (standard IHC practice). Keep staining batch, exposure to DAB, and scoring thresholds consistent; interpret differences cautiously because HPA reports low consistency between antibody staining and RNA data (standard IHC practice; HPA tissue IHC reliability).

How can I distinguish true MCU staining from an IHC artefact?

Prioritise reproducible cytoplasmic staining in intact cells, consistent with the mitochondrial location and reported tissue IHC profile (UniProt Q8NE86 subcellular location; HPA tissue IHC profile). A nuclear-only pattern, staining confined to section edges or necrosis, or signal in the no-primary control should prompt investigation before a positive call (UniProt Q8NE86 subcellular location; standard IHC practice). Check endogenous peroxidase and biotin contributions because the documented detection uses streptavidin-biotin chemistry and DAB (IHC caption A00685-1; standard IHC practice). Compare cell types within the section, but avoid treating staining intensity alone as channel activity; MCU acts within a regulated uniplex complex (UniProt Q8NE86 function and subunit).

Boster reagents

Best MCU / Calcium uniporter protein, mitochondrial IHC Antibodies

A00685-1 has IHC images from paraffin sections of mouse and rat lung and human liver and bladder cancer tissue; no IF image is supplied (A00685-1 image captions; catalog IF image list).

Real IHC data

Anti-MCU Antibody ®

Cat # A00685-1

View product →

A00685-1 is listed for IHC and has paraffin-section images from mouse and rat lung and human liver and bladder cancer tissue (catalog applications; A00685-1 image captions). It is listed as reactive with human, monkey, mouse and rat, but the supplied images do not show monkey tissue or IF/ICC (catalog reactivity; A00685-1 image captions; catalog IF image list).

Which to pick: For tissue IHC, choose A00685-1 at a starting concentration of 2–5 μg/ml in paraffin sections; its image captions report EDTA retrieval at pH 8.0, while the fixative is unreported (datasheet: 2–5 μg/ml; A00685-1 image captions). For IF/ICC, this catalog provides no validated option: A00685-1 does not list IF/ICC and has no IF image (catalog applications; catalog IF image list). For cross-species IHC, A00685-1 is listed as reactive with human, monkey, mouse and rat, with IHC images supplied for human, mouse and rat (catalog reactivity; A00685-1 image captions).

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

References

UniProt Consortium. UniProt entry Q8NE86 (MCU_HUMAN, Calcium uniporter protein, mitochondrial).
Human Protein Atlas. MCU tissue IHC expression (reliability: Approved).
Human Protein Atlas. MCU subcellular location (ICC-IF): Localized to the mitochondria..
Human Protein Atlas. MCU antibody validation summary (1 antibodies).
The Regulatory Mechanism and Biological Significance of Mitochondrial Calcium Uniporter in the Migration, Invasion, Angiogenesis and Growth of Gastric Cancer. OncoTargets and therapy 2020 — PMC7680189.
Dihydroartemisinin represses oral squamous cell carcinoma progression through downregulating mitochondrial calcium uniporter. Bioengineered 2022 — PMC8805845.
MCU That Is Transcriptionally Regulated by Nrf2 Augments Malignant Biological Behaviors in Oral Squamous Cell Carcinoma Cells. BioMed research international 2021 — PMC8195654.
Harpagide attenuates cerebral ischemic injury by modulating mitochondrial calcium homeostasis associated with the AMPK-MCU axis. Scientific reports 2026 — PMC13462545.
PubMed PMID:14702039 — UniProt-cited evidence.
PubMed PMID:15164054 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.