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NPC2 / NPC intracellular cholesterol transporter 2 · IHC design guide
Design Immunohistochemistry for NPC2
Plan NPC2 chromogenic IHC in paraffin sections using the IHC-validated antibody A01582-3 (datasheet). Use epididymal glandular cells as a high-staining reference and consider secretion when interpreting staining that differs from RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NPC2 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); lysosomal protein (UniProt) | |
| Staining pattern | Cytoplasmic in glandular cells and hepatocytes; highest in epididymis (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A01582-3) | |
| Positive control | Epididymis+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01582-3) | |
| Caveat | Secretion can make tissue staining differ from RNA expression (HPA tissue IHC) | |
| Regulation | Epididymis-enriched expression (HPA tissue IHC) | |
| Isoform / epitope | 2 isoforms; signal peptide 1–19 is absent from the mature chain (UniProt) |
Section 1
Recommended NPC2 IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet A01582-3); the three published NPC2 IHC protocols below provide tissue-specific starting points (PMC3017059; PMC3798307; PMC3406925).
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A01582-3) |
| Fixation | Image fixative and duration unreported (datasheet A01582-3); 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 A01582-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01582-3) |
| Primary antibody | Rabbit anti-NPC2, 0.5-1μg/ml (datasheet A01582-3) |
| Primary incubation | Overnight at 4 °C (datasheet A01582-3) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01582-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NPC2-positive staining in glandular cells of epididymis (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most glandular cells and in hepatocytes. Highest expression in the epididymis. No signal in the no-primary control. |
💡Decision noteFor the catalog antibody, start with heat-mediated EDTA retrieval at pH 8.0 (datasheet A01582-3). The published excerpts do not specify retrieval conditions (PMC3017059; PMC3798307; PMC3406925).
Section 2
What Is the Expected NPC2 Staining Pattern?
NPC2 should stain mainly in the cytoplasm of glandular cells and hepatocytes, with the strongest reported tissue expression in epididymal glandular cells (HPA: tissue IHC profile; High in epididymis). A lysosome-associated cytoplasmic pattern is consistent with its localisation; NPC2 is also secreted and has no transmembrane segment (UniProt P61916: localisation and topology). HPA rates the tissue staining evidence Enhanced but notes only medium consistency with RNA expression, partly because NPC2 can be secreted (HPA: tissue reliability).
| Strong cytoplasmic staining in epididymal glandular cells, with weaker staining in other expected cells. | This fits the strongest HPA result: epididymal glandular cells are High, while liver hepatocytes are Medium (HPA: tissue IHC). Assess intensity within the named cell population, rather than treating every cell in a positive tissue as equally positive (HPA: cell-specific tissue results). |
| Cytoplasmic staining in kidney distal tubules, lung macrophages or testicular Leydig cells. | Each named cell population is High in HPA tissue IHC (HPA: High in kidney distal tubules, lung macrophages and testicular Leydig cells). A cytoplasmic distribution agrees with the broader tissue profile and NPC2’s lysosomal localisation (HPA: cytoplasmic profile; UniProt P61916: lysosome). |
| Predominantly nuclear or sharply membranous staining, without the expected cytoplasmic pattern. | Treat this as a localisation mismatch requiring a specificity check, rather than confirmed NPC2 staining (HPA: cytoplasmic tissue profile; UniProt P61916: no transmembrane segment). Review a known-positive section and the negative detection control before interpreting the compartment (standard IHC practice). |
| Strong staining in a cell population reported as undetected, or widespread staining across unrelated cell types. | For example, HPA reports adipocytes and esophageal squamous epithelial cells as Not detected (HPA: tissue IHC). Unexpected staining can reflect antibody cross-reactivity or endogenous detection activity; compare its distribution with a negative detection control (standard IHC practice). |
| Diffuse staining across tissue or no staining in epididymal glandular cells. | Uniform haze can obscure the cell-specific cytoplasmic pattern (HPA: tissue IHC profile). No signal in a High reference cell population raises a sensitivity or procedure concern, but does not alone identify the failed step (HPA: High in epididymal glandular cells; standard IHC practice). |
💡Expected NPC2 appearanceCall a section positive when the named cell population shows cytoplasmic signal—ideally strong in epididymal glandular cells; predominant nuclear staining or uniform tissue-wide haze is suspect (HPA: High in epididymal glandular cells and cytoplasmic profile; UniProt P61916: lysosome; standard IHC practice).
| Cell-specific reference levels | HPA reports High staining in epididymal glandular cells, kidney distal tubules, lung macrophages and testicular Leydig cells; hepatocytes are Medium (HPA: tissue IHC). Use the named cells to judge a result. A Not detected call for gallbladder glandular cells does not contradict NPC2 detection in gallbladder bile, which is a different specimen (HPA: gallbladder glandular cells Not detected; UniProt P61916: gallbladder bile). |
| Secreted and intracellular NPC2 | NPC2 is secreted, associated with the endoplasmic reticulum and lysosome, and targeted to lysosomes through cell-surface M6PR-mediated uptake (UniProt P61916: localisation). Interpret cytoplasmic staining in the context of both intracellular trafficking and secretion; HPA cautions that tissue RNA and protein locations may differ (HPA: reliability description). |
| Processing and molecular forms | The precursor has a signal sequence at residues 1–19 and a mature chain at 20–151; two isoforms and glycosylation sites at 58 and 135 are annotated (UniProt P61916: processing, isoforms and glycosylation). The supplied record gives no antibody epitope, so it cannot establish which forms an IHC antibody detects or predict a staining difference between them. |
| Strength of IHC evidence | HPA labels CAB032888 IHC Enhanced and HPA000835 IHC Supported (HPA: antibody validation). Its tissue profile is also Enhanced, with medium antibody–RNA consistency (HPA: tissue reliability). These labels support comparison with the reported cell pattern; they do not establish an expected ICC-IF image or a target-specific fixation effect (HPA: subcellular record; supplied evidence scope). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive epididymal glandular cells have no chromogenic signal. | An absent result conflicts with the High HPA reference but does not identify the cause (HPA: epididymal glandular cells High). | Check section integrity, detection reagents, antibody dilution and antigen retrieval against the catalog antibody’s IHC-P instructions; run a positive control in the same staining batch (standard IHC practice). |
| Signal is chiefly nuclear or sharply outlines plasma membranes. | That distribution differs from the reported cytoplasmic tissue pattern and NPC2’s non-transmembrane topology (HPA: cytoplasmic profile; UniProt P61916: topology). | Compare with a known-positive section and a negative detection control; review antibody specificity before scoring the unexpected compartment (standard IHC practice). |
| Adipocytes or esophageal squamous epithelial cells stain strongly. | HPA reports these named cell populations as Not detected; cross-reactivity or endogenous detection activity is possible (HPA: tissue IHC). | Inspect a negative detection control and compare the signal with staining in the expected positive cells; do not call the whole tissue positive from an unrelated cell population (standard IHC practice; HPA: cell-specific results). |
| Diffuse chromogen obscures cell boundaries throughout the section. | Background can hide the cell-specific cytoplasmic distribution used to interpret NPC2 (HPA: tissue IHC profile; standard IHC practice). | Review blocking, washes, antibody dilution and detection-control staining using the catalog antibody’s IHC-P instructions (standard IHC practice). |
| A low or absent signal appears in an HPA negative-reference cell population. | A Not detected HPA call applies to its named cells and assay context; it is not a blanket absence claim for every cell in that tissue (HPA: tissue IHC). | Score the specified cell population, and include a High reference population in the staining run before deciding whether the result is informative (HPA: tissue IHC; standard IHC practice). |
| Can ICC-IF be used to confirm the subcellular pattern? | HPA lists NPC2 as Secreted but provides no main ICC-IF location or cell-line images (HPA: subcellular record). | Use the separate IF/ICC guide for that application. Do not treat the tissue IHC pattern as a validated ICC-IF image; the supplied HPA record provides none (HPA: tissue IHC and subcellular record). |
Experiment evidence
IHC & IF images showing NPC2 staining
A01582-3 · IHCIHC analysis of NPC2 using anti-NPC2 antibody (A01582-3). NPC2 was detected in paraffin-embedded section of human tonsil tissue. Heat mediat
A01582-3 · IHCIHC analysis of NPC2 using anti-NPC2 antibody (A01582-3). NPC2 was detected in paraffin-embedded section of mouse spleen tissue. Heat mediat
A01582-3 · IFIF analysis of NPC2 using anti-NPC2 antibody (A01582-3). NPC2 was detected in immunocytochemical section of A431 cells. Enzyme antigen retri
A01582-2 · IHCIHC analysis of NPC2 using anti-NPC2 antibody (A01582-2). NPC2 was detected in a paraffin-embedded section of human renal cancer tissue. Hea
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000119655-NPC2/tissue/cerebral+cortex#imid_20903735
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000119655-NPC2/tissue/colon#imid_20903653
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000119655-NPC2/tissue/liver#imid_20903654
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000119655-NPC2/tissue/kidney#imid_20903647
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NPC2 IHC & IF
🧪Run kidney first: distal tubules should stain strongly (HPA: High in kidney distal tubules). Use adipose tissue adipocytes as the negative comparison (HPA: Not detected in adipocytes); on the kidney slide, other structures should show no more than control-level background, though HPA does not establish them as NPC2-negative.
Positive control tissue: Epididymis (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for NPC2; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) control, a rabbit isotype control matched to the primary antibody’s class and clonality, and NPC2-knockout tissue or validated immunizing-peptide competition as a biological negative (standard IHC control practice; caption: rabbit anti-NPC2). Block endogenous peroxidase and check for endogenous biotin in kidney before interpreting the biotin-based DAB signal (caption: biotinylated secondary, SABC and DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A01582-3 paraffin-section caption does not state a fixative (caption: fixative not stated). The demonstrated IHC workflow uses heat retrieval in EDTA at pH 8.0; its dependence on retrieval has not been established by a comparison (caption: EDTA heat retrieval). Frozen-section and IF performance cannot be ranked from the supplied evidence, and endogenous kidney biotin may complicate the caption’s biotin-based detection (HPA: kidney distal tubules High; caption: SABC; standard IHC practice).
HPA tissue IHC evidence for NPC2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex.). 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 |
|---|---|---|---|---|
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Distal tubules | High | Protein (IHC) | HPA → |
| Lung | Macrophages | High | Protein (IHC) | HPA → |
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cerebral cortex | Endothelial cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NPC2 IHC Tips
Troubleshoot NPC2 staining in paraffin sections by checking retrieval, cellular pattern and detection controls against the documented IHC result.
What should I change if NPC2 staining is weak after antigen retrieval?
Start with heat-mediated retrieval in EDTA pH 8.0 for paraffin sections (datasheet A01582-3). The documented tonsil stain then used 10% goat serum blocking and 1 μg/ml primary antibody overnight at 4°C (datasheet A01582-3). If signal is weak, verify heating, cooling and buffer pH across the run before changing antibody concentration; compare a known positive control such as epididymal glandular cells (HPA: High in epididymis glandular cells). If retrieval conditions are changed, keep a section under the documented conditions alongside it, because differences in staining alone do not establish a biological difference in NPC2 abundance.
Can the documented NPC2 staining tell me which fixation conditions work best?
The NPC2 tonsil example identifies a paraffin section but does not state its fixative, so target-specific fixation sensitivity is unknown (datasheet A01582-3: fixative not stated). Record fixative, fixation duration and processing history for each specimen before comparing chromogenic intensity, because these variables can affect antigen preservation and access in routine IHC. Use sections prepared under the same conditions for an optimization series, with the documented EDTA pH 8.0 retrieval as the starting point (datasheet A01582-3). Include a positive control in each run; a loss of signal after a fixation change cannot be assigned to NPC2 without controlling retrieval and detection.
Which staining pattern is plausible for NPC2 in paraffin sections?
Assess NPC2 primarily as cytoplasmic staining within the relevant cells: it is a soluble protein associated with lysosomes and the endoplasmic reticulum and can be secreted (UniProt P61916: subcellular location and topology). A granular intracellular pattern is plausible for lysosomal signal, while a clean plasma-membrane rim would need independent validation because NPC2 has no transmembrane segment (UniProt P61916: topology). Compare the pattern with expected cell populations, such as kidney distal tubules or lung macrophages (HPA: High in kidney distal tubules and lung macrophages). Secreted NPC2 may complicate attribution of extracellular staining to its producing cell (UniProt P61916: secreted); inspect cellular morphology and controls before scoring it.
Could processing or isoforms explain a change in NPC2 IHC staining?
NPC2 has 2 annotated isoforms, a signal peptide at residues 1–19, and a mature chain spanning 20–151 (UniProt P61916: isoforms and processing). It also has glycosylation sites at residues 58 and 135, which are relevant when assessing where an antibody epitope lies (UniProt P61916: glycosylation). The supplied IHC caption does not identify the antibody epitope or demonstrate isoform discrimination (datasheet A01582-3: tonsil IHC caption). Check the catalog antibody’s immunogen or epitope documentation before interpreting staining changes as altered processing or isoform expression, and keep retrieval and detection conditions matched across sections.
How should I follow up NPC2 IHC with multiplex immunofluorescence?
Use IF as a separate validation experiment: the supplied NPC2 IHC example documents paraffin-section chromogenic staining, while the HPA subcellular record lists no ICC/IF images (datasheet A01582-3; HPA: subcellular record). Pair NPC2 with a validated marker of the expected cell population, such as a macrophage marker in lung, where macrophages show high NPC2 staining (HPA: High in lung macrophages). Choose fluorophores after inspecting unstained tissue for autofluorescence, and include single-stain controls to assess bleed-through. Because NPC2 is soluble and lysosomal rather than transmembrane (UniProt P61916: location and topology), optimize permeabilisation to reach the intracellular epitope while preserving punctate structure; its antibody epitope is unspecified (datasheet A01582-3: tonsil IHC caption).
How can I distinguish NPC2 signal from chromogenic background?
First inspect a section without primary antibody to identify signal from the secondary reagent, endogenous enzyme activity or tissue pigment in the chromogenic workflow. The documented assay used a biotinylated secondary, avidin–biotin complex and DAB, so a detection-control section is especially useful when staining is diffuse (datasheet A01582-3). Maintain appropriate peroxidase blocking for DAB detection and check for endogenous biotin contribution when using avidin–biotin chemistry; these are general IHC controls. Compare cell-associated staining with an expected positive population, such as kidney distal tubules (HPA: High in kidney distal tubules), and assess whether signal persists at section edges or damaged areas.
What is a defensible way to quantify NPC2 chromogenic staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because NPC2 can be intracellular or secreted (UniProt P61916: subcellular location). Within a consistent region, record the percentage of positive target cells and staining intensity, or calculate an H-score from the same predefined intensity categories for every section. Normalize positive counts to the number of eligible cells, or report positive-cell density per mm² of viable tissue when area is the sampling unit. Keep retrieval, detection, exposure to DAB and image analysis settings consistent; stratify distinct populations such as kidney distal tubules and lung macrophages rather than pooling them (HPA: High in kidney distal tubules and lung macrophages).
When should an apparent NPC2-positive IHC result be questioned?
A credible result should fit both the cell type and a plausible intracellular or secreted distribution: NPC2 is lysosomal, endoplasmic-reticulum associated and secreted (UniProt P61916: subcellular location). Strong staining confined to a plasma-membrane rim is unexpected for a protein without a transmembrane segment (UniProt P61916: topology). Compare suspect staining with expected high-signal populations, such as epididymal glandular cells or lung macrophages (HPA: High in epididymis glandular cells and lung macrophages). Treat edge-restricted colour, necrotic regions or staining retained without primary antibody as potential artefacts; control for endogenous peroxidase and avidin–biotin detection background before assigning those signals to NPC2 (datasheet A01582-3: DAB and avidin–biotin detection).
Boster reagents
Best NPC2 / NPC intracellular cholesterol transporter 2 IHC Antibodies
The catalog shows anti-NPC2 IHC data from paraffin sections of human tonsil, human renal cancer, and mouse spleen, plus IF data from A431 cells (catalog image captions).
Real IHC data
Anti-Niemann Pick C2/NPC2 ® Antibody
Cat # A01582-3
Real IHC data
Anti-Niemann Pick C2/NPC2 Antibody ®
Cat # A01582-2
A01582-3 has paraffin-section IHC images from human tonsil and mouse spleen and an IF/ICC image from A431 cells (A01582-3 image captions). A01582-2 has a paraffin-section IHC image from human renal cancer tissue (A01582-2 image caption).
Which to pick: For human tissue IHC, both are polyclonal options: A01582-3 has a human tonsil example at 1 μg/ml, while A01582-2 has a human renal cancer example at 2 μg/ml (catalog clonality; respective IHC captions). Choose A01582-3 for IF/ICC or mouse IHC because it lists those applications or reactivity and shows A431-cell IF and mouse spleen IHC (A01582-3 catalog applications/reactivity and image captions). Both IHC captions describe paraffin sections; neither reports the fixative (A01582-2 and A01582-3 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P61916 (NPC2_HUMAN, NPC intracellular cholesterol transporter 2).
- Human Protein Atlas. NPC2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. NPC2 subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. NPC2 antibody validation summary (2 antibodies).
- A novel role for Niemann-Pick disease type 2C protein in papillae formation. PloS one 2011 — PMC3017059.
- Characterization of Niemann-Pick Type C2 protein expression in multiple cancers using a novel NPC2 monoclonal antibody. PloS one 2013 — PMC3798307.
- The role of NPC2 gene in glioma was investigated based on bioinformatics analysis. Scientific reports 2024 — PMC11333723.
- Lung cancer signatures in plasma based on proteome profiling of mouse tumor models. Cancer cell 2011 — PMC3406925.
- PubMed PMID:8418812 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:12508121 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NPC2 (NPC intracellular cholesterol transporter 2). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); lysosomal protein (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A01582-3). Validated IHC antibody: A01582-3 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Epididymis (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A01582-3); 2. Fixation: Image fixative and duration unreported (datasheet A01582-3); 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 A01582-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01582-3); 8. Primary antibody: Rabbit anti-NPC2, 0.5-1μg/ml (datasheet A01582-3); 9. Primary incubation: Overnight at 4 °C (datasheet A01582-3). Watch for: Fixation: Keep paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01582-3); Caveat: Secretion can make tissue staining differ from RNA expression (HPA tissue IHC); Isoform / epitope: 2 isoforms; signal peptide 1–19 is absent from the mature chain (UniProt); Regulation: Epididymis-enriched expression (HPA tissue IHC). Evidence: UniProt P61916, HPA tissue IHC · Enhanced, HPA ICC-IF · Secreted, Datasheet A01582-3, PMC3017059, PMC3798307. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NPC2/immunohistochemistry-ihc-guide.