MCU / Calcium uniporter protein, mitochondrial · IHC design guide

Design Immunohistochemistry for MCU

Plan MCU staining in paraffin sections using the cytoplasmic tissue pattern reported by HPA (HPA tissue IHC). The guide pairs that observation with MCU’s inner mitochondrial membrane location (UniProt) and the catalog antibody’s IHC dilution of 2–5 μg/ml (datasheet A00685-1).

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 Cytoplasmic staining in most tissues (HPA tissue IHC), antibody A00685-1, validated IHC image, and IHC protocol steps — Printable **MCU** IHC protocol sheet — expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC), 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	Cytoplasmic staining in most tissues (HPA tissue IHC)
	Staining pattern	Cytoplasmic stain; high in several glandular cell types (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 paraffin-section fixation consistent across samples (standard IHC practice; not target-specific)
	Caveat	Staining and RNA expression show low consistency (HPA tissue IHC)
	Regulation	No expression regulator annotated (UniProt)
	Isoform / epitope	3 isoforms; epitope coverage is unspecified (UniProt)

Section 1

Recommended MCU IHC & IF Protocols

The catalog antibody’s IHC-P protocol is followed by three published MCU staining protocols for paraffin sections (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 retrieval at pH 8.0 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 is an inner mitochondrial membrane protein with two transmembrane segments (UniProt Q8NE86 topology). In paraffin sections, expect cytoplasmic staining in many cell types, including strong staining in selected glandular cells and neurons (HPA tissue IHC). HPA rates its tissue staining Approved but reports low consistency with RNA expression; interpret intensity alongside controls rather than assuming every stained cell has high MCU expression (HPA tissue IHC).

What am I looking at on my slide?
Cytoplasmic staining in adrenal, appendix, breast, or colon glandular cells.	This matches the High staining reported for those cell populations (HPA tissue IHC). A granular pattern is compatible with mitochondrial localisation (UniProt Q8NE86; HPA subcellular ICC-IF), but chromogenic IHC may not resolve individual mitochondria (general IHC practice).
Predominantly nuclear staining, or a sharp cell-surface outline without cytoplasmic signal.	These patterns conflict with MCU's inner mitochondrial membrane location (UniProt Q8NE86 topology). Check counterstain and chromogen distribution, then compare an appropriate control before calling the pattern specific (general IHC practice).
Strong staining confined to cells expected to stain weakly, while an expected High population is blank.	Consider cross-reactivity or endogenous detection activity (general IHC practice). HPA reports Low staining in liver cholangiocytes and soft-tissue fibroblasts, but neither is a proven negative control; cell identity and staining conditions still need checking (HPA tissue IHC).
Diffuse staining across tissue, extracellular spaces, and areas without clear cell boundaries.	This distribution is difficult to reconcile with cytoplasmic MCU staining (HPA tissue IHC; UniProt Q8NE86). Review background in reagent controls and check blocking, washes, and chromogen development (general IHC practice).
No detectable staining in a documented High population.	A blank result in colon glandular cells or cerebral-cortex neurons conflicts with the reported tissue pattern (HPA tissue IHC). First verify tissue identity and control performance; then assess retrieval, antibody conditions, and detection as general IHC variables.

💡Expected MCU appearanceCall a result positive when identifiable cells show predominantly cytoplasmic staining, potentially granular, with strong signal in an HPA High population such as colon glandular cells; isolated nuclear or extracellular colour is suspect (HPA tissue IHC; UniProt Q8NE86; general IHC practice).

How each factor affects the staining
Cell population and comparator selection	HPA reports High staining in bronchial basal cells, caudate neurons, cerebellar granular-layer cells, and several glandular populations (HPA tissue IHC). Its Low categories are relative comparators, not validated negatives; choose the same cell population when comparing runs.
Antibody evidence and interpretation limit	HPA016480 is IHC Approved, while HPA notes low consistency between antibody staining and RNA expression (HPA antibody validation; HPA tissue IHC). This supports using the reported pattern as a guide, with additional controls for an unexpected result.
Topology and epitope accessibility	MCU has two membrane-spanning segments and matrix-facing regions on either side of a short intermembrane segment (UniProt Q8NE86 topology). Without an antibody epitope or target-specific retrieval study, these facts do not predict which retrieval condition will work best.
Processing and isoforms	UniProt lists a 51–351 mature chain and three isoforms (UniProt Q8NE86). Epitope coverage across the isoforms is unspecified here; avoid attributing a staining difference to processing or an isoform without antibody-specific evidence.
IF/ICC Q&A: what localisation should be expected?	Mitochondrial signal is expected in IF/ICC; HPA supports that location and lists images from A-431, U-251MG, and U2OS (HPA subcellular ICC-IF). That observation helps assess localisation but does not establish an IHC-P staining condition.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Expected High cells are blank, and the run control is also blank.	The run may have a detection or reagent failure (general IHC practice). HPA's High calls describe observed staining, not guaranteed intensity in every section (HPA tissue IHC).	Check reagent order, expiry, and detection-control performance; repeat with a documented High population in the same run (general IHC practice; HPA tissue IHC).
Expected High cells are blank, but the run control stains.	Section identity, retrieval, antibody conditions, or local tissue preservation may differ (general IHC practice). No MCU-specific fixation sensitivity is reported in this payload.	Confirm the cell population, then compare controlled retrieval and antibody conditions using the same detection system (general IHC practice).
Colour is concentrated in nuclei rather than cytoplasm.	The compartment conflicts with the mitochondrial location assigned to MCU (UniProt Q8NE86; HPA subcellular ICC-IF); counterstain or nonspecific signal may complicate reading (general IHC practice).	Inspect the chromogen and counterstain separately, compare controls, and require cytoplasmic staining before assigning MCU positivity (general IHC practice; HPA tissue IHC).
Weakly rated cells stain strongly while expected High cells do not.	Cross-reactivity or endogenous detection activity is possible (general IHC practice). HPA's Low designations are observations, not proof that those cells lack MCU (HPA tissue IHC).	Verify cell identity, examine reagent controls, and repeat alongside an HPA High comparator before interpreting the discrepancy (general IHC practice; HPA tissue IHC).
Broad, hazy colour obscures individual cells.	Background from blocking, washing, or excessive development can impair interpretation (general IHC practice); it does not establish broader MCU distribution.	Review reagent controls and adjust blocking, washes, or development within the established IHC workflow (general IHC practice). Score only identifiable cellular staining.
Staining is cytoplasmic but lacks visible mitochondrial puncta.	Chromogenic sections may not resolve mitochondria individually (general IHC practice). HPA reports cytoplasmic tissue staining, while IF/ICC supports mitochondrial localisation (HPA tissue IHC; HPA subcellular ICC-IF).	Assess whether the expected cell populations stain and whether controls are clean. Do not reject an otherwise consistent IHC result solely for lacking visible puncta (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

🧪Run appendix first and score its glandular cells, which show High MCU staining (HPA: High in appendix glandular cells). HPA detects MCU in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and judge non-glandular cells on the same slide against those controls without assuming they are MCU-negative (HPA: no negative tissue listed; 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: Include a no-primary, secondary-only slide, a matched rabbit IgG isotype control, and MCU-knockout material as a biological specificity control (selected IHC caption: rabbit anti-MCU antibody; standard IHC practice). Block endogenous peroxidase and check inflammatory cells for residual chromogen signal in appendix, particularly with the caption’s biotin-based DAB detection (HPA: appendix tissue; selected IHC caption: biotinylated secondary, SABC and DAB; standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A00685-1 paraffin-section caption does not state its fixative (selected IHC caption: fixative not stated). That caption uses heat retrieval in EDTA at pH 8.0 before chromogenic detection; treat this as a starting condition, since retrieval dependence has not been established (selected IHC caption: EDTA pH 8.0). Frozen sections and IF are not established as easier by the supplied evidence; appendix inflammatory cells may produce endogenous peroxidase signal that needs control-based interpretation (HPA: appendix tissue; standard IHC practice).

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 MCU staining in paraffin sections by checking retrieval, staining controls, mitochondrial localisation and cell type before comparing chromogenic signal across samples.

Which retrieval conditions should I start with for MCU in paraffin sections?

Start with heat-mediated antigen retrieval in EDTA buffer at pH 8.0 (datasheet A00685-1). The selected mouse lung paraffin-section image used this retrieval before staining with 2 μg/mL catalog antibody overnight at 4°C (caption A00685-1). If signal is weak, compare retrieval duration on matched sections while keeping antibody concentration and detection conditions fixed (standard IHC practice). Include a section processed without primary antibody to distinguish retrieval-related background from MCU staining (standard IHC practice). Assess whether improved signal retains the expected mitochondrial distribution, since MCU resides in the mitochondrial inner membrane (UniProt Q8NE86 localisation).

How should I troubleshoot fixation-related changes in MCU staining?

The selected image documents a paraffin section but does not state its fixative, so MCU-specific fixation sensitivity is unknown (caption A00685-1). Record the fixative and fixation duration for each specimen, then compare matched sections processed with the same EDTA pH 8.0 retrieval (standard IHC practice; datasheet A00685-1). If staining differs, examine morphology and no-primary controls before attributing the difference to antigen loss (standard IHC practice). Keep antibody incubation consistent at the documented 2 μg/mL overnight at 4°C during that comparison (caption A00685-1). Tissue staining patterns and MCU topology do not establish a target-specific fixation effect (HPA tissue IHC; UniProt Q8NE86 topology).

What MCU staining pattern is plausible in chromogenic tissue sections?

Expect cytoplasmic staining consistent with mitochondrial localisation, because MCU is an inner mitochondrial membrane protein (UniProt Q8NE86 localisation; HPA tissue IHC profile). At sufficient resolution, a granular cytoplasmic pattern is more persuasive than uniformly nuclear staining (UniProt Q8NE86 localisation; standard IHC interpretation). HPA reports cytoplasmic expression in most tissues and high staining in bronchial basal cells, among other cell populations (HPA tissue IHC). Compare signal within anatomically matched cells and include a no-primary control before calling diffuse DAB deposition specific (standard IHC practice). Chromogenic resolution cannot establish which side of the inner membrane an antibody recognises; epitope mapping is needed for that claim (UniProt Q8NE86 topology; standard IHC interpretation).

Could MCU isoforms or epitope position explain discordant IHC results?

MCU has 3 annotated isoforms, but the supplied product evidence does not identify this antibody’s epitope or isoform coverage (UniProt Q8NE86 isoforms; caption A00685-1). The mature annotated chain spans residues 51–351, with transmembrane segments at 234–255 and 263–284 (UniProt Q8NE86 processing and topology). Matrix-facing and intermembrane-facing regions therefore pose different epitope-access questions that cannot be resolved from the tissue image alone (UniProt Q8NE86 topology; caption A00685-1). Compare matched sections under the documented EDTA pH 8.0 retrieval before assigning staining differences to isoforms (datasheet A00685-1; standard IHC practice). Interpret any suspected epitope effect cautiously because residue 97 can be glutathionylated, but its relationship to this antibody is unknown (UniProt Q8NE86 modified residues; caption A00685-1).

How can IF help investigate an ambiguous MCU IHC pattern?

Use IF as a separate corroborating experiment, since HPA supports mitochondrial MCU localisation in cell images (HPA subcellular). Multiplex MCU with a mitochondrial marker and a marker defining the expected cell population, then assess overlap within individual cells (UniProt Q8NE86 localisation; standard IF practice). Choose fluorophores in channels with low measured tissue autofluorescence and include unstained and single-colour controls (standard IF practice). Optimise permeabilisation against the antibody’s mapped epitope: MCU has matrix-facing regions at 51–233 and 285–351, and an intermembrane-facing region at 256–262 (UniProt Q8NE86 topology). Because this antibody’s epitope is unspecified, IF colocalisation supports localisation but cannot identify the recognised membrane face (caption A00685-1; UniProt Q8NE86 topology).

How do I reduce diffuse or patchy DAB background in MCU IHC?

First inspect a no-primary section and the tissue edge to separate detection-system deposition from cellular staining (standard IHC practice). The selected protocol used 10% goat serum blocking, biotinylated secondary antibody and streptavidin–biotin detection with DAB (caption A00685-1). Check the peroxidase block and, where appropriate, controls for endogenous biotin before changing the MCU primary concentration (standard IHC practice). Compare matched sections with shorter chromogen development and adequate washes while retaining the documented 2 μg/mL primary incubation initially (standard IHC practice; caption A00685-1). Accept a staining change as improvement only if cellular signal remains compatible with mitochondrial MCU and background falls in controls (UniProt Q8NE86 localisation; standard IHC practice).

What should I measure when comparing MCU IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION

Score MCU in a predefined cell population using percentage of positive cells and staining intensity, or report an H-score with its scoring bins stated (standard IHC quantification). Normalise counts or positive area to the number of eligible cells or analysed tissue area, rather than to the entire section (standard IHC quantification). Keep EDTA retrieval at pH 8.0, antibody concentration at 2 μg/mL, and chromogen development consistent across compared slides (datasheet A00685-1; caption A00685-1; standard IHC practice). Exclude folds, necrotic regions and edges using prespecified rules (standard IHC quantification). Report the cell compartment scored, since MCU is mitochondrial and HPA describes broadly cytoplasmic tissue staining (UniProt Q8NE86 localisation; HPA tissue IHC profile).

When should an apparent MCU-positive IHC result be questioned?

Question predominantly nuclear, extracellular or edge-restricted DAB signal because MCU is an inner mitochondrial membrane protein (UniProt Q8NE86 localisation; standard IHC interpretation). Check whether staining sits in the intended cell population; HPA reports high signal in bronchial basal cells but low signal in oral mucosal squamous epithelial cells (HPA tissue IHC). Exclude necrotic areas and compare no-primary and peroxidase-block controls when brown signal follows damaged tissue or endogenous enzyme activity (standard IHC practice). Interpret intensity differences cautiously because HPA rates its tissue staining Approved while noting low consistency with RNA expression (HPA tissue IHC reliability). Stronger chromogenic staining alone does not establish greater mitochondrial calcium transport, which also depends on uniporter regulatory subunits (UniProt Q8NE86 function and subunit).

Boster reagents

Best MCU / Calcium uniporter protein, mitochondrial IHC Antibodies

A00685-1 has real IHC images from paraffin sections of mouse and rat lung and human liver and bladder cancer tissue (catalog IHC captions). No IF images are provided (catalog IF images).

Real IHC data

Anti-MCU Antibody ®

Cat # A00685-1

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A00685-1 shows IHC staining in paraffin sections of mouse and rat lung and human liver and bladder cancer tissue (catalog IHC captions). Its listed applications include IHC but exclude IF/ICC; its listed reactivity is human, monkey, mouse, and rat (catalog applications and reactivity).

Which to pick: Choose A00685-1 for paraffin-section tissue IHC, starting within its 2–5 μg/ml IHC range (catalog IHC captions; datasheet: 2–5 μg/ml); the fixative is unreported (catalog IHC captions). No IF/ICC antibody can be recommended from this payload because A00685-1 has no listed IF/ICC application or IF image (catalog applications; catalog IF images). For work across species, A00685-1 has IHC images for human, mouse, and rat tissue, while monkey is listed only under reactivity; it is rabbit hosted, and its clonality is unreported (catalog IHC captions; catalog reactivity; catalog host and clone).

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.