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ORAI1 / Calcium release-activated calcium channel protein 1 · IHC design guide
Design Immunohistochemistry for ORAI1
Plan chromogenic ORAI1 IHC in paraffin sections using skin keratinocytes as a high staining reference (HPA tissue IHC). Interpret the observed cytoplasmic staining alongside ORAI1’s annotated cell membrane location (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ORAI1 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 observed; cell membrane annotated (HPA tissue IHC; UniProt) | |
| Staining pattern | Strong keratinocyte cytoplasm; cytoplasmic in most tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Skin+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 fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Cytoplasmic IHC can complicate membrane interpretation (HPA tissue IHC; UniProt) | |
| Regulation | Similar in naive and effector T helper cells (UniProt) | |
| Isoform / epitope | 2 isoforms; check whether the epitope is extracellular or cytoplasmic (UniProt) |
Section 1
Recommended ORAI1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by four published ORAI1 chromogenic IHC protocols (PMC8739746; PMC3871607; PMC5190020; PMC5342032).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A00909-1); 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-ORAI1, 10 μg/mL (datasheet A00909-1) |
| 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 | ORAI1-positive staining in keratinocytes of skin (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues, highly abundant in skin. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen-retrieval setting); adapt to the tissue and antibody used.
Section 2
What Is the Expected ORAI1 Staining Pattern?
ORAI1 is a four-pass cell-membrane protein, with basolateral localisation also annotated (UniProt Q96D31 topology and subcellular location). In paraffin-section IHC, expect the clearest staining in skin keratinocytes and testicular Leydig cells (HPA: High in both). HPA also reports cytoplasmic staining in most tissues. Its tissue IHC assessment is Approved, with medium consistency against RNA data and external verification pending (HPA: tissue IHC reliability).
| Keratinocytes or Leyig cells show strong staining, with a membrane-associated component. | These are the best-supported positive cell populations (HPA: High in skin keratinocytes and testis Leydig cells). Membrane localisation agrees with ORAI1 topology (UniProt Q96D31). Assess the cells themselves; colour in adjacent spaces does not establish ORAI1 expression (standard IHC practice). |
| Positive cells show cytoplasmic staining without a sharp membrane outline. | This can agree with the reported tissue-IHC profile, which describes cytoplasmic expression in most tissues (HPA: tissue IHC profile). Record compartment and cell type separately. A membrane outline is biologically plausible, but should not be imposed as the sole acceptable paraffin-IHC pattern (UniProt Q96D31 subcellular location). |
| Signal is predominantly nuclear or appears outside cells. | Neither location matches the annotated cell-membrane protein or the reported cytoplasmic tissue-IHC profile (UniProt Q96D31 subcellular location; HPA: tissue IHC profile). Treat it as suspect until section morphology, counterstain, and a detection-only control have been checked (standard IHC practice). |
| Strong signal appears in adipocytes or glomerular cells. | Those sampled cell populations were not detected in HPA tissue IHC (HPA: adipose adipocytes; kidney glomerular cells). Consider cross-reactivity or endogenous detection activity, especially if a no-primary control also stains (standard IHC practice). A discrepant slide alone cannot identify which cause applies. |
| Colour covers broad regions or known-positive cells remain unstained. | Uniform colour obscures cell-specific interpretation (standard IHC practice). Absence of signal in skin keratinocytes or Leydig cells conflicts with HPA's High observations, but does not by itself prove that ORAI1 is absent (HPA: skin; testis). Check controls and slide quality before scoring. |
💡Expected ORAI1 appearanceA convincing positive is strong, cell-associated staining in skin keratinocytes or testicular Leydig cells, with a membrane-associated and/or cytoplasmic pattern (HPA: High in both; HPA: cytoplasmic tissue profile; UniProt Q96D31 subcellular location); isolated nuclear colour or strong staining of HPA-undetected adipocytes is suspect (HPA: adipocytes Not detected; standard IHC practice).
| Compartment and assay | UniProt places ORAI1 at the cell membrane, including basolateral membrane, while HPA tissue IHC describes cytoplasmic staining (UniProt Q96D31; HPA: tissue IHC profile). Interpret a paraffin section against both observations and its cell morphology; neither source establishes that every positive cell must show a crisp membrane rim. |
| Antibody epitope and topology | ORAI1 has four transmembrane segments with extracellular and cytoplasmic loops and termini (UniProt Q96D31 topology). The supplied record does not locate the IHC antibody's epitope, so it cannot predict which retrieval or permeabilisation condition exposes that epitope. Optimize those steps empirically with tissue controls (standard IHC practice). |
| Cell state and puncta | After calcium-store depletion, ORAI1 can colocalise with STIM1 in membrane puncta at ER–plasma-membrane junctions (UniProt Q96D31 subcellular location). The supplied HPA paraffin-IHC profile does not establish puncta as an expected tissue-section pattern; do not require them to call an ordinary section positive. |
| Isoforms and tissue expression | UniProt lists alpha and beta isoforms and protein expression in naive CD4 and CD8 T cells (UniProt Q96D31). HPA reports hematopoietic cells in bone marrow as Not detected in its tissue-IHC samples (HPA: bone marrow). Those observations concern different specimens and methods; the supplied antibody data do not establish isoform discrimination. |
| IF/ICC Q&A: what localisation is supported? | HPA summarises ORAI1 as membrane-localised in ICC-IF, but supplies no main-location assignment or cell-line images here (HPA: subcellular ICC-IF). Use that as a limited localisation cross-check; this section provides no IF/ICC protocol or cell-line-specific expected intensity. |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in skin keratinocytes or Leydig cells. | A failed run is possible because HPA observed High staining in these cells (HPA: skin; testis); absence alone does not isolate the failed step. | Check tissue preservation, positive-control performance, primary-antibody step, detection reagents, and counterstain; adjust retrieval or antibody concentration with controls (standard IHC practice). |
| All tissue compartments develop similar diffuse colour. | The distribution lacks the cell specificity needed to interpret HPA's keratinocyte and Leydig-cell observations (HPA: skin; testis). Excess background is possible (standard IHC practice). | Inspect the no-primary control; review blocking, washes, detection time, and primary concentration using the same tissue batch (standard IHC practice). |
| Signal is concentrated in nuclei. | Predominantly nuclear staining conflicts with UniProt's membrane localisation and HPA's cytoplasmic tissue profile (UniProt Q96D31; HPA: tissue IHC profile). | Compare with morphology and counterstain, then repeat with a detection-only control and a known-positive tissue (standard IHC practice). |
| Adipocytes or glomerular cells stain strongly. | HPA lists those cell populations as Not detected; cross-reactivity or endogenous detection activity may account for the discrepancy (HPA: adipose; kidney; standard IHC practice). | Check a no-primary control, inspect which cells carry signal, and compare with positive skin or testis on the same run (HPA: skin; testis; standard IHC practice). |
| Cytoplasmic colour is present but the membrane rim is weak. | HPA describes cytoplasmic tissue-IHC expression despite UniProt's membrane annotation (HPA: tissue IHC profile; UniProt Q96D31). The contrast is not sufficient evidence of assay failure. | Score cell type, intensity, and compartment separately; compare with HPA-positive cells and confirm that the no-primary control is clear (HPA: skin; testis; standard IHC practice). |
| Bone-marrow cells are negative despite expected T-cell expression. | UniProt reports ORAI1 protein in naive T cells, while HPA's sampled bone-marrow hematopoietic cells were Not detected by tissue IHC (UniProt Q96D31 tissue specificity; HPA: bone marrow). | Do not use bone-marrow negativity alone to reject the run. Verify staining in skin or testis and interpret any T-cell result in its specimen and assay context (HPA: skin; testis; standard IHC practice). |
Experiment evidence
IHC & IF images showing ORAI1 staining
A00909-1 · IHCImmunohistochemistry of ORAI1 in human ovary tissue with ORAI1 antibody at 2.5 μg/ml.
A00909-1 · IHCImmunohistochemistry of ORAI1 in human spleen tissue with ORAI1 antibody at 10 μg/mL.
A00909-1 · IFImmunofluorescence of ORAI1 in Human Spleen cells with ORAI1 antibody at 20 μg/mL.
M00909 · IHCImmunohistochemistry of ORAI1 in human spleen tissue with ORAI1 antibody at 5 μg/mL.
M00909 · IHCImmunohistochemistry of ORAI1 in human spleen tissue with ORAI1 antibody at 2.5 μg/mL.
M00909 · IFImmunofluorescence of ORAI1 in human spleen tissue with ORAI1 antibody at 20 μg/ml.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000276045-ORAI1/tissue/cerebral+cortex#imid_4203468
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000276045-ORAI1/tissue/colon#imid_4203402
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000276045-ORAI1/tissue/liver#imid_4203403
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000276045-ORAI1/tissue/kidney#imid_4203396
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ORAI1 IHC & IF
🧪Run skin first: keratinocytes should stain (HPA: High in skin keratinocytes). Use adipose tissue as the negative comparator, scoring adipocytes (HPA: Not detected in adipocytes); on the skin slide, assess background in morphologically distinct cells that lack staining, if present, without assuming a particular cell type is ORAI1-negative.
Positive control tissue: Skin (Keratinocytes, 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 ORAI1; derive a cell-line control from the positive tissue's cell type (Keratinocytes) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) section, a concentration-matched isotype control appropriate to the primary antibody’s host species and clonality, and ORAI1 knockout tissue or a peptide-block control if the immunizing peptide is available. For chromogenic skin IHC, quench endogenous peroxidase and distinguish native pigment from the reaction product (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and the effect of antigen retrieval are unreported in the supplied evidence; the selected A00909-1 ovary IHC caption also leaves the fixative unreported (selected-SKU caption). The evidence does not establish whether frozen sections or IF would be easier than paraffin IHC. In skin, native pigment can complicate chromogenic scoring (standard IHC practice).
HPA tissue IHC evidence for ORAI1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Skin | Keratinocytes | High | Protein (IHC) | HPA → |
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Medium | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ORAI1 IHC Tips
Troubleshoot ORAI1 chromogenic IHC in paraffin sections using matched controls, compartment aware scoring and the antibody’s documented tissue evidence (UniProt Q96D31; HPA; caption A00909-1).
What should I change when ORAI1 staining is weak after antigen retrieval?
Use citrate buffer at pH 6.0 for HIER at 95–98 °C for 20 min before chromogenic IHC (page retrieval rule: cytoplasmic/membrane antigen). ORAI1 has 4 transmembrane segments, so assess membrane and cytoplasmic staining separately after retrieval (UniProt Q96D31 topology; HPA: cytoplasmic tissue staining). If signal remains weak, compare a fallback retrieval condition on serial sections while holding antibody concentration and DAB development constant (standard IHC practice). Include a no-primary control with each condition to identify retrieval associated background (standard IHC practice). Compare the same cell population across conditions, since tissue composition can otherwise mimic a gain in signal (standard IHC practice).
Could fixation explain inconsistent ORAI1 staining between paraffin blocks?
ORAI1-specific fixation sensitivity is unknown from the supplied evidence; the ovary IHC caption reports no fixative (caption A00909-1). Record fixative, fixation duration and section age for each block before attributing differences to ORAI1 abundance (standard IHC practice). If processing changed, stain matched control sections together using citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval rule). Keep antibody incubation and DAB development identical, and assess a no-primary section for processing related background (standard IHC practice). Do not infer a fixation effect from ORAI1 topology or phosphosites without a direct comparison (UniProt Q96D31 topology/PTMs).
How should I interpret diffuse cytoplasmic ORAI1 staining when I expect a membrane channel?
ORAI1 is annotated at the cell membrane, including the basolateral membrane, while tissue IHC reports cytoplasmic expression in most tissues (UniProt Q96D31 localisation; HPA: tissue profile). Score crisp membrane staining separately from diffuse cytoplasmic staining, using the same criteria across sections (standard IHC practice). Store depletion can concentrate ORAI1 with STIM1 in membrane puncta, but routine chromogenic sections do not establish that functional state (UniProt Q96D31 localisation). Compare staining with a no-primary section and with the expected cell population before calling diffuse signal specific (standard IHC practice; HPA: High in skin keratinocytes). Avoid treating every cytoplasmic deposit as membrane channel localisation (UniProt Q96D31 localisation).
Can this IHC assay distinguish ORAI1 alpha from beta or identify an exposed epitope?
ORAI1 has 2 reported isoforms, alpha and beta, but the supplied IHC caption does not map the antibody epitope or establish isoform selectivity (UniProt Q96D31 isoforms; caption A00909-1). Check the catalog antibody’s immunogen or epitope documentation before making either claim (standard IHC practice). ORAI1 has cytoplasmic regions at residues 1–87, 141–173 and 256–301, with extracellular loops at 106–119 and 195–234 (UniProt Q96D31 topology). Interpret staining only as ORAI1 compatible until isoform discrimination is independently demonstrated (UniProt Q96D31 isoforms; standard IHC practice). Keep retrieval and detection identical when comparing tissues, since protocol changes can alter epitope accessibility (standard IHC practice).
How can I assess ORAI1 by IF alongside a cell type marker?
Treat IF/ICC as a separate application: the ovary chromogenic IHC caption does not establish IF performance (caption A00909-1). In skin, multiplex with a keratinocyte marker and check whether ORAI1 signal falls in the expected cells (HPA: High in skin keratinocytes; standard IF practice). Select a spectrally separated, preferably far red fluorophore after inspecting unstained tissue for autofluorescence (standard IF practice). Choose permeabilisation according to the documented antibody epitope: intracellular ORAI1 regions require access, whereas an extracellular loop may be tested without permeabilisation (UniProt Q96D31 topology; standard IF practice). Include single stain and no-primary controls to assess bleed through and background before interpreting apparent colocalisation (standard IF practice).
What causes widespread brown signal in ORAI1 chromogenic IHC?
Start with a no-primary section to identify staining from detection reagents, endogenous activity or tissue pigment (standard IHC practice). For peroxidase and DAB detection, apply an endogenous peroxidase block and compare its effect under otherwise matched conditions (standard IHC practice). If background persists, optimise blocking, antibody concentration, wash steps and DAB development one variable at a time (standard IHC practice). Inspect cell boundaries and section edges before assigning diffuse deposits to ORAI1; its annotated localisation includes the plasma membrane (UniProt Q96D31 localisation). Use the same exposure and scoring criteria across sections, and compare against a documented positive cell population (standard IHC practice; HPA: High in skin keratinocytes).
How should I quantify ORAI1 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because membrane localisation and cytoplasmic tissue staining are both reported for ORAI1 (UniProt Q96D31 localisation; HPA: tissue profile). Record the percentage of positive cells and an H-score from 0–300, using consistent intensity thresholds across batches (standard IHC practice). For sparse populations, report positive cells per mm² of viable analysed tissue and state the sampled area (standard IHC practice). Normalise cell based results to the number of eligible cells, excluding necrosis, folds and section edges by a prespecified rule (standard IHC practice). Analyse matched positive and no-primary controls alongside each batch to detect staining drift (standard IHC practice).
When is an ORAI1 positive result more likely to be artefact?
A credible result follows reproducible cell and compartment patterns: ORAI1 is membrane associated, while HPA tissue IHC also describes cytoplasmic staining (UniProt Q96D31 localisation; HPA: tissue profile). Compare candidate signal with expected positive cells, such as skin keratinocytes, and a no-primary section (HPA: High in skin keratinocytes; standard IHC practice). Staining confined to section edges, folds or necrotic areas warrants caution because those regions can produce misleading deposits (standard IHC practice). Brown signal that persists without primary antibody may reflect endogenous peroxidase or detection background rather than ORAI1 (standard IHC practice). Report membrane and cytoplasmic scores separately rather than equating either pattern alone with channel activity (UniProt Q96D31 localisation; standard IHC practice).
Boster reagents
Best ORAI1 / Calcium release-activated calcium channel protein 1 IHC Antibodies
Two anti-ORAI1 antibodies have human tissue IHC images and human IF images (catalog captions); both list Human and Mouse reactivity, while M00909 also lists Rat (catalog reactivity).
Real IHC data
Anti-ORAI1 Antibody
Cat # A00909-1
Real IHC data
Anti-ORAI1 Monoclonal Antibody [3F6H5]
Cat # M00909
A00909-1 lists IHC-P and IF, with IHC shown in human ovary and IF shown in human spleen cells (catalog applications and image captions). M00909 lists IHC-P and IF, with both images from human spleen samples (catalog applications and image captions).
Which to pick: For tissue IHC, choose A00909-1 for the human ovary example or M00909 for the human spleen example; both list IHC-P, but their image captions do not report the fixative (catalog applications and IHC image captions). For IF, both list IF and show human spleen samples; neither payload establishes a separate ICC validation (catalog applications and IF image captions). Choose M00909 when Rat reactivity or a mouse monoclonal is needed; A00909-1 is a rabbit antibody listing Human and Mouse reactivity (catalog reactivity, host and clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q96D31 (ORAI1_HUMAN, Calcium release-activated calcium channel protein 1).
- Human Protein Atlas. ORAI1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ORAI1 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. ORAI1 antibody validation summary (1 antibodies).
- Expression of Orai1 and STIM1 in human oral squamous cell carcinogenesis. Journal of dental sciences 2022 — PMC8739746.
- Antibody-mediated targeting of the Orai1 calcium channel inhibits T cell function. PloS one 2013 — PMC3871607.
- Orai1 promotes tumor progression by enhancing cancer stemness via NFAT signaling in oral/oropharyngeal squamous cell carcinoma. Oncotarget 2016 — PMC5190020.
- FGF4 induces epithelial-mesenchymal transition by inducing store-operated calcium entry in lung adenocarcinoma. Oncotarget 2016 — PMC5342032.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16147976 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ORAI1 (Calcium release-activated calcium channel protein 1). Expected localisation Cytoplasm observed; cell membrane annotated (HPA tissue IHC; UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A00909-1 at 10 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Skin (Keratinocytes); negative Adipose tissue. 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 A00909-1); 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-ORAI1, 10 μg/mL (datasheet A00909-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Cytoplasmic IHC can complicate membrane interpretation (HPA tissue IHC; UniProt); Isoform / epitope: 2 isoforms; check whether the epitope is extracellular or cytoplasmic (UniProt); Regulation: Similar in naive and effector T helper cells (UniProt). Evidence: UniProt Q96D31, HPA tissue IHC · Approved, HPA ICC-IF · Membrane, Datasheet A00909-1, PMC8739746, PMC3871607. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ORAI1/immunohistochemistry-ihc-guide.