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NPC1 / NPC intracellular cholesterol transporter 1 · IHC design guide
Design Immunohistochemistry for NPC1
Plan chromogenic NPC1 tissue IHC with catalog antibody A00428, starting at 2.5 μg/mL (datasheet: A00428). Expect generally cytoplasmic tissue staining (HPA tissue IHC), while NPC1 resides in late endosome and lysosome membranes (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NPC1 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 IHC (HPA tissue IHC); late endosome/lysosome membrane (UniProt) | |
| Staining pattern | High staining in kidney tubules; generally cytoplasmic (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| 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 | IHC staining has medium consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Expression regulation not annotated (UniProt) | |
| Isoform / epitope | 2 isoforms; check whether the epitope is lumenal or cytoplasmic (UniProt) |
Section 1
Recommended NPC1 IHC & IF Protocols
The catalog antibody's IHC-P protocol is accompanied by two published NPC1 IHC protocols (PMC11961396; PMC12292827).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A00428); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-NPC1, 2.5 μg/mL (datasheet A00428) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NPC1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval). If staining is weak, evaluate the published Tris-EDTA pH 9 method (PMC12292827).
Section 2
What Is the Expected NPC1 Staining Pattern?
NPC1 is a 13-pass protein of late-endosome and lysosome membranes, so positive cells should show cytoplasmic staining consistent with those compartments (UniProt O15118 topology and subcellular location). HPA reports general cytoplasmic IHC expression, with high staining in adrenal glandular cells, kidney tubular cells, ovarian follicle cells, seminal-vesicle glandular cells, and cells in testicular seminiferous ducts (HPA tissue IHC). Its tissue IHC reliability is Approved, with medium agreement between staining and RNA data (HPA tissue IHC).
| Cytoplasmic staining in kidney tubular cells or adrenal glandular cells, with little nuclear signal. | This fits HPA's high staining in those cell types and its general cytoplasmic profile (HPA tissue IHC). A punctate or granular appearance would be consistent with late-endosome and lysosome membranes, but HPA does not establish a required granule pattern in these sections (UniProt O15118 subcellular location; HPA tissue IHC). |
| Strong staining confined mainly to nuclei, plasma membranes, or extracellular material. | That distribution conflicts with the expected late-endosome and lysosome membrane location and HPA's cytoplasmic IHC profile (UniProt O15118 subcellular location; HPA tissue IHC). Treat it as a possible staining artefact; assess section morphology and controls before assigning NPC1 positivity (general IHC practice). |
| Intense staining in cells outside the expected stained population, while the expected cells remain faint. | For a chosen tissue, compare the stained cells with HPA's cell-level observations; for example, kidney staining is reported as high in tubular cells (HPA tissue IHC). An unexpected cell distribution can reflect cross-reactivity or endogenous detection activity (general IHC practice). It is not definitive evidence against NPC1 because HPA reports low tissue specificity and no negative tissues (HPA tissue IHC). |
| A diffuse stain covers many cell types, lumina, or section-free areas without a clear cytoplasmic pattern. | This obscures the general cytoplasmic profile reported by HPA (HPA tissue IHC). Background outside cells suggests nonspecific reagent or detection signal; compare a no-primary control and inspect wash and blocking conditions (general IHC practice). Uniform brown color alone does not establish NPC1-positive cells (general IHC practice). |
| No detectable staining in kidney tubular cells or adrenal glandular cells. | Both are reported as high-staining cell populations, so an absent result warrants a technical check (HPA tissue IHC). Check section integrity, retrieval, antibody conditions, and detection controls using the IHC-validated antibody's instructions (general IHC practice). HPA's Approved rating has medium staining–RNA agreement and does not guarantee positivity in every specimen (HPA tissue IHC). |
💡Expected NPC1 appearanceCall a section positive when appropriate cells show discernible cytoplasmic staining, especially the HPA high-staining populations; isolated nuclear, extracellular, or section-wide diffuse color is suspect (HPA tissue IHC; UniProt O15118 subcellular location; general IHC practice).
| Cell population and tissue | HPA reports high staining in five specified cell populations, medium staining in appendix glandular, bone-marrow hematopoietic, and bronchial respiratory epithelial cells, and low staining in several others (HPA tissue IHC). Choose a documented high-staining population for a positive control; low staining alone is a weak technical benchmark (HPA tissue IHC; general IHC practice). |
| Antibody validation | HPA lists HPA026618 and CAB070132 as IHC Approved; neither is listed as IHC Enhanced in the supplied record (HPA antibodies). HPA describes overall tissue IHC reliability as Approved with medium staining–RNA consistency, so interpret unexpected patterns with controls (HPA tissue IHC; general IHC practice). |
| Membrane topology and epitope position | NPC1 has 13 transmembrane segments and lumenal and cytoplasmic regions (UniProt O15118 topology). The antibody epitope is not supplied, so topology cannot identify which region the antibody detects or predict a target-specific retrieval requirement (UniProt O15118 topology; supplied antibody record). |
| Processing, glycosylation, and isoforms | UniProt lists a signal peptide at residues 1–22, a mature chain beginning at residue 23, 19 glycosylation sites, and two isoforms (UniProt O15118 processing, glycosylation, isoforms). Without an epitope or isoform-specific antibody record, these features do not justify predicting different tissue staining or fixation sensitivity (supplied antibody record). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known high-staining cells are blank. | The assay may have failed, although HPA's reported high level does not guarantee every specimen will stain (HPA tissue IHC; general IHC practice). | Run a documented high-staining tissue alongside the sample; check section preservation, the antibody's IHC-P instructions, retrieval and detection steps, and reagent controls (HPA tissue IHC; general IHC practice). |
| Color appears mainly in nuclei or extracellular spaces. | This conflicts with NPC1's late-endosome and lysosome membrane location and HPA's cytoplasmic profile (UniProt O15118 subcellular location; HPA tissue IHC). | Review morphology and a no-primary control; adjust nonspecific staining controls before scoring the compartment as NPC1 positive (general IHC practice). |
| Unexpected cells stain more strongly than the documented population. | Cross-reactivity or endogenous detection activity is possible, but NPC1 also has low tissue specificity and HPA supplies no negative-tissue list (general IHC practice; HPA tissue IHC). | Compare cell identities with the HPA tissue profile; include no-primary and detection controls, and use an independently validated antibody if the assignment remains uncertain (HPA tissue IHC; HPA antibodies; general IHC practice). |
| The whole section has weak, diffuse color. | Background from antibody or detection reagents may obscure cellular staining (general IHC practice). | Inspect a no-primary control, washing and blocking; score cells only where staining can be distinguished from section-wide background (general IHC practice). |
| A low-staining tissue gives little or no signal. | HPA reports low staining in several specified cell populations; weak signal there is a poor standalone test of assay performance (HPA tissue IHC). | Evaluate a documented high-staining population in parallel before changing assay conditions, and avoid calling the low-staining sample NPC1 negative on that result alone (HPA tissue IHC; general IHC practice). |
| IF/ICC: what pattern should be expected? | UniProt places NPC1 on late-endosome and lysosome membranes, but HPA provides only a Membrane summary, no main ICC-IF location, and no cell-line images (UniProt O15118 subcellular location; HPA subcellular ICC-IF). | Assess intracellular membrane-associated signal against appropriate IF controls; treat a specific punctate pattern as a hypothesis, not an HPA-verified ICC-IF finding (UniProt O15118 subcellular location; HPA subcellular ICC-IF; general IF practice). |
Experiment evidence
IHC & IF images showing NPC1 staining
A00428 · IHCImmunohistochemistry of NPC1 in mouse kidney tissue with NPC1 antibody at 2.5 μg/mL.
A00428 · IFImmunofluorescence of NPC1 in Mouse Kidney cells with NPC1 antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000141458-NPC1/tissue/cerebral+cortex#imid_18724812
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000141458-NPC1/tissue/colon#imid_18729334
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000141458-NPC1/tissue/liver#imid_18729343
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000141458-NPC1/tissue/kidney#imid_18729336
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NPC1 IHC & IF
🧪Run kidney first; tubular cells should stain (HPA: High in kidney cells in tubules). HPA detects NPC1 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and judge cells outside the stained tubules against background without treating them as proven NPC1-negative cells (HPA: no negative tissue rows).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: NPC1 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 carries no ICC-IF cell line for NPC1; 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 and an isotype control matched to the primary antibody’s host species and immunoglobulin class; use NPC1 knockout tissue as a biological negative (standard IHC controls). In kidney sections, block endogenous peroxidase for chromogenic detection and check endogenous biotin if using avidin–biotin detection (standard kidney IHC practice).
⚠️Feasibility: A target-specific fixation window and the effect of antigen retrieval are unreported in the supplied evidence; the mouse-kidney IHC caption does not state a fixative (A00428 tissue-IHC caption: fixative not stated). The evidence does not establish whether frozen sections or IF are easier than paraffin IHC (supplied application evidence: IHC caption only; HPA: no ICC-IF image cell lines). Kidney tubules can show endogenous biotin signal with avidin–biotin detection, so confirm the detection background on the control slide (standard kidney IHC practice).
HPA tissue IHC evidence for NPC1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium 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 → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Ovary | Follicle cells | High | Protein (IHC) | HPA → |
| Seminal vesicle | Glandular cells | High | Protein (IHC) | HPA → |
| Testis | Cells in seminiferous ducts | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: NPC1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced NPC1 IHC Tips
Troubleshoot NPC1 staining in paraffin sections by checking retrieval, compartmental pattern, controls, and scoring before interpreting biological differences.
What retrieval conditions should I try first for weak NPC1 staining in paraffin sections?
Start with citrate buffer at pH 6.0 for heat-induced antigen retrieval at 95–98 °C for 20 minutes (page retrieval setting). Keep section thickness, heating vessel, cooling time, and the IHC-validated antibody concentration constant while comparing staining across runs (standard IHC practice). If staining remains weak, test a more alkaline retrieval buffer on adjacent sections as a fallback, with a matched positive control and an assessment of tissue integrity (standard IHC practice). Score improvement by specific cytoplasmic puncta rather than stronger diffuse color, because NPC1 resides on late endosome and lysosome membranes (UniProt O15118 localisation).
How should I troubleshoot weak NPC1 staining when fixation details are missing?
NPC1-specific sensitivity to fixative type or duration is unknown from the supplied evidence; the mouse kidney caption reports an antibody concentration of 2.5 µg/mL but does not state a fixative (A00428 tissue-IHC caption). Record the fixative, fixation interval, processing schedule, section age, and retrieval conditions for each paraffin block before comparing staining (standard IHC practice). If material permits, stain freshly cut sections from blocks processed under documented, consistent conditions alongside the affected sections (standard IHC practice). Interpret any difference as a processing-associated observation until matched specimens establish which step changed antigen detection (standard IHC practice).
What NPC1 staining pattern is plausible in chromogenic tissue IHC?
Expect predominantly cytoplasmic, often punctate staining consistent with late endosome and lysosome membranes; NPC1 is an integral membrane protein with 13 transmembrane segments (UniProt O15118 localisation and topology). At light-microscope resolution, chromogenic IHC usually cannot prove that individual puncta are lysosomes, so describe their distribution without assigning every deposit to an organelle (standard IHC practice). Kidney tubular cells provide a documented high-staining reference, and the selected antibody has a mouse kidney tissue-IHC image (HPA: High in kidney tubules; A00428 tissue-IHC caption). Treat uniform nuclear staining or a crisp plasma-membrane-only pattern as a reason to recheck specificity and detection controls (UniProt O15118 localisation; standard IHC practice).
How can NPC1 topology and isoforms affect interpretation of a negative stain?
NPC1 has 2 annotated isoforms, 13 transmembrane segments, and several lumenal and cytoplasmic regions (UniProt O15118 isoforms and topology). Its record also lists 19 glycosylation sites, while the supplied evidence does not identify the catalog antibody’s epitope or establish equal recognition of both isoforms (UniProt O15118 glycosylation; supplied antibody evidence). Therefore, a negative paraffin-section stain cannot by itself distinguish absent protein from an inaccessible or altered epitope (standard IHC interpretation). Compare adjacent sections under controlled retrieval conditions and, where available, use an independently validated antibody against a disclosed region before making an isoform-specific claim (standard IHC practice).
How should I plan IF follow-up to investigate NPC1-positive cells and puncta?
For a separate IF experiment, pair NPC1 with a validated marker of the cell type being examined, and compare the channels with the chromogenic pattern in matched tissue regions (standard IF practice). The supplied tissue profile reports high staining in kidney tubular cells, which can guide cell identification but does not validate this antibody for IF (HPA: High in kidney tubules; A00428 tissue-IHC caption). Choose fluorophores after checking the tissue’s autofluorescence in unstained sections, and include single-channel controls for multiplex acquisition (standard IF practice). Optimise permeabilisation against a known epitope location: NPC1 exposes both lumenal and cytoplasmic regions, while this antibody’s epitope is unspecified (UniProt O15118 topology; supplied antibody evidence).
What should I check when NPC1 IHC has widespread brown background?
Compare a no-primary control with the complete stain to identify signal from the chromogenic detection system, and inspect unstained tissue for pigment (standard IHC practice). Block endogenous peroxidase before DAB development, then check whether nonspecific secondary binding, inadequate washing, or excessive primary concentration explains residual color (standard IHC practice). The selected mouse kidney image used 2.5 µg/mL, but that caption does not establish an optimal concentration for other specimens or processing conditions (A00428 tissue-IHC caption). Judge any adjustment against expected cytoplasmic puncta and a matched positive tissue control, since NPC1 localises to late endosome and lysosome membranes (UniProt O15118 localisation; standard IHC practice).
How should I quantify NPC1 IHC across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and tissue compartment before examining group labels, then keep section thickness, imaging, and threshold settings consistent (standard IHC practice). Report the percentage of positive cells and an intensity-weighted H-score, or count NPC1-positive cells per mm² when density is the relevant endpoint (standard IHC practice). Normalise scores to the number of evaluable cells or viable tissue area within the same annotated compartment, and exclude folds, edges, and necrotic regions (standard IHC practice). Analyse comparable cell populations because high staining is reported in kidney tubular cells and several other specified cell types, while overall RNA tissue specificity is low (HPA tissue IHC and RNA specificity).
How do I distinguish convincing NPC1 staining from tissue or detection artefact?
Give greatest weight to reproducible cytoplasmic puncta in intact cells, consistent with NPC1 on late endosome and lysosome membranes (UniProt O15118 localisation). Check whether the positive cell type is plausible for that tissue: kidney tubular cells, adrenal glandular cells, and ovarian follicle cells have reported high staining (HPA tissue IHC). Distrust signal confined to section edges, folds, necrosis, or a no-primary control, and investigate endogenous peroxidase when brown color persists after omitting primary antibody (standard IHC practice). Diffuse nuclear or plasma-membrane-only staining warrants review of morphology, retrieval, and controls before assigning it to NPC1 (UniProt O15118 localisation; standard IHC practice).
Boster reagents
Best NPC1 / NPC intracellular cholesterol transporter 1 IHC Antibodies
A00428 has IHC data from mouse kidney tissue and IF data from mouse kidney cells; its listed reactivity covers human and mouse (catalog image captions; catalog reactivity).
Real IHC data
Anti-NPC1 Antibody
Cat # A00428
Only A00428 will render a card: its IHC figure shows mouse kidney tissue stained at 2.5 μg/mL (catalog IHC image caption). Its IF figure shows mouse kidney cells at 20 μg/mL (catalog IF image caption).
Which to pick: For paraffin-section IHC, choose A00428: IHC-P is listed, and its own IHC figure shows mouse kidney tissue staining; the caption does not report the fixative (catalog applications; catalog IHC image caption). For IF with figure evidence, choose A00428; for ICC, M00428-1 lists ICC/IF and is a rabbit monoclonal, but has no supplied figure (catalog IF image caption; catalog applications; catalog host/clone; catalog image alts). For rat samples, M00428-1 lists rat reactivity, while A00428 lists human and mouse (catalog reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O15118 (NPC1_HUMAN, NPC intracellular cholesterol transporter 1).
- Human Protein Atlas. NPC1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. NPC1 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. NPC1 antibody validation summary (2 antibodies).
- NPC1 promotes the progression of hepatocellular carcinoma by mediating the accumulation of neutrophils into the tumor microenvironment. FEBS open bio 2025 — PMC11961396.
- The Niemann-Pick C1 Protein of Patients with Hepatocellular Carcinoma Is Associated with Survival Time in Males and Tumor Size in Females. Biomedicines 2025 — PMC12292827.
- GCase and LIMP2 Abnormalities in the Liver of Niemann Pick Type C Mice. International journal of molecular sciences 2021 — PMC7959463.
- Astrocyte-only Npc1 reduces neuronal cholesterol and triples life span of Npc1-/- mice. Journal of neuroscience research 2008 — PMC2634300.
- PubMed PMID:9211849 — UniProt-cited evidence.
- PubMed PMID:10425213 — UniProt-cited evidence.
- PubMed PMID:11754101 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NPC1 (NPC intracellular cholesterol transporter 1). Expected localisation Cytoplasmic tissue IHC (HPA tissue IHC); late endosome/lysosome membrane (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A00428 at 2.5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A00428); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-NPC1, 2.5 μg/mL (datasheet A00428); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: IHC staining has medium consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 2 isoforms; check whether the epitope is lumenal or cytoplasmic (UniProt); Regulation: Expression regulation not annotated (UniProt). Evidence: UniProt O15118, HPA tissue IHC · Approved, HPA ICC-IF · Membrane, Datasheet A00428, PMC11961396, PMC12292827. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NPC1/immunohistochemistry-ihc-guide.