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BCAS4 / Breast carcinoma-amplified sequence 4 · IHC design guide
Design Immunohistochemistry for BCAS4
Plan BCAS4 chromogenic IHC in paraffin sections using the general cytoplasmic tissue pattern as a reference (HPA tissue IHC). Start the catalog antibody at 5 μg/mL (datasheet), and interpret staining in light of HPA's uncertain tissue IHC reliability (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
BCAS4 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) | |
| Staining pattern | General cytoplasmic staining, including glandular cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Appendix+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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Low agreement between antibody staining and RNA (HPA tissue IHC) | |
| Regulation | Elevated in most breast cancer cell lines (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended BCAS4 IHC & IF Protocols
Compare the catalog antibody's IHC-P protocol with the published BCAS4 workflow for paraffin-embedded HCC and adjacent non-tumor sections (PMC13520631).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A13963-1); 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-BCAS4, 5 μg/mL (datasheet A13963-1) |
| 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 | BCAS4-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen retrieval); the paper does not specify retrieval conditions (PMC13520631).
Section 2
What Is the Expected BCAS4 Staining Pattern?
BCAS4 is a cytoplasmic protein with no annotated transmembrane segment (UniProt Q8TDM0). In paraffin-section IHC, look for cytoplasmic staining in glandular cells of the appendix, cervix, epididymis and gallbladder, among other cell types reported as High by HPA (HPA: tissue IHC). Treat this as a provisional pattern: HPA rates tissue IHC reliability Uncertain because antibody staining has low consistency with RNA expression (HPA: tissue IHC reliability).
| Clear cytoplasmic chromogen in appendix glandular cells, with nearby tissue structures remaining distinguishable (HPA: High in appendix glandular cells). | This fits the reported compartment and cell type (UniProt Q8TDM0: cytoplasm; HPA: tissue IHC). Record intensity and the fraction of cells stained, while retaining the Uncertain tissue IHC reliability caveat (HPA: tissue IHC reliability). |
| Strong staining is chiefly nuclear or outlines plasma membranes, with little cytoplasmic signal. | That distribution conflicts with the annotated cytoplasm and lack of a transmembrane segment (UniProt Q8TDM0). Check staining controls and section quality before interpreting it as BCAS4; HPA's tissue IHC pattern is Uncertain (HPA: tissue IHC reliability). |
| Fibroblasts stain more strongly than the glandular cells in the same section. | HPA reports Low staining in soft-tissue fibroblasts and High staining in several glandular cell populations (HPA: tissue IHC). The reversal raises possible cross-reactivity or endogenous detection activity; cell type alone cannot establish its cause (general IHC interpretation). |
| Pale or granular color extends across stroma, empty spaces and multiple cell types without a clear cytoplasmic boundary. | Treat this as diffuse background rather than a scored positive pattern (general IHC interpretation). A no-primary control can help assess detection-system background, while tissue morphology helps distinguish cellular signal from deposited chromogen (general IHC practice). |
| No staining appears in appendix glandular cells on a slide selected as a positive comparison (HPA: High in appendix glandular cells). | The result does not reproduce HPA's reported pattern; check the run and the actual cells present before calling the specimen negative (general IHC practice). HPA rates its tissue IHC evidence Uncertain, so this tissue is a comparison, not an infallible positive control (HPA: tissue IHC reliability). |
💡Expected BCAS4 appearanceCall the result provisionally positive when identifiable HPA-reported High cell populations show distinct cytoplasmic staining; dominant nuclear or membrane staining, or color outside cells, is suspicious (UniProt Q8TDM0: cytoplasm and no transmembrane segment; HPA: tissue IHC, reliability Uncertain; general IHC interpretation).
| Cell population and tissue | HPA reports High staining in appendix, cervix, epididymis and gallbladder glandular cells; esophageal squamous cells; heart cardiomyocytes; nasopharyngeal respiratory epithelium; and ovarian follicle cells (HPA: tissue IHC). Compare like cell types rather than treating an entire section as uniformly positive (general IHC interpretation). |
| Lower-staining comparisons | Soft-tissue fibroblasts, lymph-node germinal-center cells and bone-marrow hematopoietic cells are reported Low (HPA: tissue IHC). They can help assess contrast, but a Low designation does not promise an entirely blank cell population (HPA: tissue IHC; general IHC interpretation). |
| Confidence in the IHC pattern | The tissue IHC profile is Uncertain because staining and RNA expression show low consistency, and antibody HPA038812 has an Uncertain IHC assessment (HPA: tissue IHC reliability; HPA: antibody validation). Interpret a matching stain as supportive, with controls and morphology, rather than definitive proof of specificity (general IHC interpretation). |
| Protein features | BCAS4 has three annotated isoforms, no signal peptide, no propeptide, no transmembrane segment and no annotated glycosylation sites (UniProt Q8TDM0). The supplied record gives no antibody epitope, so it cannot establish which isoforms this stain detects or predict antigen-retrieval sensitivity. |
| IF/ICC Q&A: where should signal appear? | HPA reports approved cytosol and intermediate-filament locations in ICC-IF images from A-431, MCF-7 and U2OS, and rates HPA038812 ICC Approved (HPA: subcellular ICC-IF; HPA: antibody validation). This answers the localisation question for IF/ICC; it does not establish an IF protocol or override the Uncertain tissue IHC assessment (HPA: antibody validation). |
| Situation | Likely cause | Next action |
|---|---|---|
| A reported High cell population has no visible signal (HPA: tissue IHC). | The staining run or tissue-cell selection may be at issue; HPA's Uncertain IHC profile also limits certainty about the expected signal (HPA: tissue IHC reliability; general IHC practice). | Confirm that the named cells are present, inspect the run's controls, and review the validated IHC procedure used for the antibody (general IHC practice). |
| Nuclei or membranes dominate the stain. | This conflicts with BCAS4's cytoplasmic annotation and absence of a transmembrane segment (UniProt Q8TDM0); the stain may be nonspecific (general IHC interpretation). | Compare matched positive and low-staining cell populations, then check detection controls before assigning BCAS4 localisation (HPA: tissue IHC; general IHC practice). |
| Fibroblasts or germinal-center cells stain strongly (HPA: Low in these cells). | The pattern departs from HPA's reported Low staining and may reflect cross-reactivity or detection background; the slide alone cannot distinguish them (HPA: tissue IHC; general IHC interpretation). | Review staining without primary antibody and compare cytoplasmic signal in HPA-reported High cells on a suitable section (HPA: tissue IHC; general IHC practice). |
| Diffuse color obscures cell boundaries. | Excess background or chromogen deposition can prevent reliable compartment scoring (general IHC practice). | Check the no-primary control, blocking and washing steps, then score only cells whose cytoplasm can be distinguished from surrounding color (general IHC practice). |
| Signal appears in the no-primary control. | The detection system or endogenous tissue activity may produce color independently of primary-antibody binding (general chromogenic IHC practice). | Review the detection chemistry and its appropriate blocking control before attributing matched-slide staining to BCAS4 (general chromogenic IHC practice). |
| A tissue stains, but its pattern disagrees with HPA's listed cell populations. | HPA rates tissue IHC Uncertain because staining and RNA expression have low consistency; antibody HPA038812 is also IHC Uncertain (HPA: tissue IHC reliability; HPA: antibody validation). | Document the tissue, cell type, compartment, intensity and controls; report the discrepancy without treating either a single slide or the HPA pattern as conclusive (general IHC interpretation). |
Experiment evidence
IHC & IF images showing BCAS4 staining
A13963-1 · IHCImmunohistochemistry of BCAS4 in human breast carcinoma with BCAS4 antibody at 5 μg/mL.
A13963-1 · IFImmunofluorescence of BCAS4 in human breast carcinoma tissue with BCAS4 antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000124243-BCAS4/tissue/cerebral+cortex#imid_10873571
HPA · StomachHuman Protein Atlas IHC, Stomach. Source: https://www.proteinatlas.org/ENSG00000124243-BCAS4/tissue/stomach+2#imid_10873431
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000124243-BCAS4/tissue/colon#imid_10873481
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000124243-BCAS4/tissue/liver#imid_10873498
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA038812; green = BCAS4, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA038812; green = BCAS4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for BCAS4 IHC & IF
🧪Run appendix first and expect glandular cells to stain (HPA: High in appendix glandular cells). HPA detects BCAS4 in all 45 scored tissues, so use no-primary and isotype controls for the negative comparison; cells on the positive slide that lack specific signal should remain at background (HPA: no negative tissue listed; standard IHC practice).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: None in HPA: BCAS4 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 BCAS4 in A-431, MCF-7, U2OS, with annotated localisation: Intermediate filaments (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and either host- and isotype-matched IgG for a monoclonal primary or host-matched nonimmune IgG for a polyclonal primary; use a BCAS4 knockout sample as a biological negative when available (standard IHC practice). For chromogenic detection, quench endogenous peroxidase and block endogenous biotin if using biotin-based detection (standard IHC practice).
⚠️Feasibility: A BCAS4-specific fixation window and antigen-retrieval requirement are unreported in the supplied evidence; the A13963-1 breast-carcinoma IHC caption gives 5 µg/mL but does not state the fixative (selected-SKU caption). Frozen sections are not established as easier, while ICC-IF images exist for A-431, MCF-7 and U2OS and show cytosol and intermediate-filament localization (HPA subcellular). In appendix, check that apparent glandular staining is cellular rather than chromogen trapped in luminal mucus (standard IHC practice).
HPA tissue IHC evidence for BCAS4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: BCAS4 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced BCAS4 IHC Tips
Troubleshoot BCAS4 staining in paraffin section chromogenic IHC, with a focused IF/ICC comparison (page application; UniProt Q8TDM0).
How should I adjust retrieval when BCAS4 staining is weak or uneven?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min for BCAS4 paraffin section IHC (page IHC retrieval rule). Run a known staining section and a no primary control alongside the test section, keeping section thickness and detection conditions matched (standard IHC practice). If staining remains weak, compare a carefully controlled longer retrieval or an alternative buffer on adjacent sections, while watching for tissue damage and increased background (standard IHC practice). Judge improvement by cleaner cytoplasmic staining, since BCAS4 is annotated in the cytoplasm, rather than by stronger staining alone (UniProt Q8TDM0 subcellular location).
Could fixation explain weak BCAS4 staining in paraffin sections?
BCAS4 specific sensitivity to fixative type or fixation duration is unknown from the supplied evidence; the selected tissue image caption does not state a fixative (A13963-1 tissue IHC caption). Record the fixative, fixation duration, tissue size and processing history before comparing sections, because these variables can change antigen accessibility in paraffin IHC (standard IHC practice). Compare similarly processed sections using citrate pH 6.0 retrieval at 95–98 °C for 20 min, with the same antibody and detection conditions (page IHC retrieval rule; standard IHC practice). Treat any improvement after changing fixation as an empirical finding for those specimens, and confirm it against morphology and controls (standard IHC practice).
What BCAS4 staining pattern should I accept in tissue sections?
Assess BCAS4 primarily as a cytoplasmic signal, since UniProt assigns it to the cytoplasm and HPA describes general cytoplasmic tissue expression (UniProt Q8TDM0 subcellular location; HPA tissue IHC profile). HPA IF places BCAS4 in the cytosol and intermediate filaments, but that finer pattern may be difficult to resolve with chromogenic tissue IHC (HPA subcellular; standard IHC practice). Examine glandular cells in appendix or cervix as documented high staining examples, while retaining the same processing and scoring rules across sections (HPA tissue IHC: High in appendix and cervix glandular cells; standard IHC practice). Investigate predominantly nuclear or sharply membranous signal with controls before assigning it to BCAS4 (UniProt Q8TDM0 subcellular location and topology; standard IHC practice).
Can isoforms or epitope accessibility change the IHC result?
BCAS4 has 3 annotated isoforms, so establish which sequence region the catalog antibody recognizes before comparing specimens or published stains (UniProt Q8TDM0 isoforms; standard IHC practice). No epitope position is supplied here, and the record lists no transmembrane segment, signal peptide, glycosylation site or modified residue that would identify an epitope specific processing step (UniProt Q8TDM0 topology, processing and PTMs). Keep citrate pH 6.0, 95–98 °C, 20 min retrieval constant while evaluating antibody concentration or exposure (page IHC retrieval rule; standard IHC practice). If staining differs, seek isoform and epitope documentation before attributing the difference to alternative splicing (UniProt Q8TDM0 isoforms; standard IHC practice).
How should I compare a BCAS4 IF result with chromogenic IHC?
For secondary IF/ICC work, pair BCAS4 with a validated glandular epithelial marker when examining glandular cells, and check whether the signals occur in the expected cells (HPA tissue IHC: High in appendix and cervix glandular cells; standard IF practice). Choose a red or far red fluorophore after checking tissue autofluorescence in an unstained control, and acquire single channel controls before interpreting overlap (standard IF practice). Because BCAS4 is cytoplasmic with no transmembrane segment, test mild permeabilisation for access to a cytosolic epitope; the catalog antibody’s epitope position is unspecified (UniProt Q8TDM0 subcellular location and topology; standard IF practice). Compare compartment and cell identity with IHC, without assuming the two detection methods yield identical intensity (standard IHC/IF practice).
How can I reduce diffuse brown background without losing BCAS4 signal?
First compare the stained slide with no primary and detection only controls to locate background from the chromogen or detection system (standard IHC practice). Apply a peroxidase block before HRP and DAB development, then check whether brown deposits persist in the control; these are general chromogenic IHC steps, not BCAS4 specific evidence (standard IHC practice). Optimise blocking, washing and catalog antibody concentration on adjacent sections, using the caption’s 5 µg/mL as a documented tissue image condition rather than a universal working concentration (A13963-1 tissue IHC caption; standard IHC practice). Preserve cytoplasmic contrast and tissue morphology when judging a change, since BCAS4 has a cytoplasmic annotation (UniProt Q8TDM0 subcellular location; standard IHC practice).
How should I score BCAS4 across differently cellular tissue regions? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, and count cytoplasmic staining rather than all brown pixels because BCAS4 is annotated in the cytoplasm (UniProt Q8TDM0 subcellular location; standard IHC practice). For a selected population, report percent positive cells and an H-score based on intensity classes 0–3, or report positive cell density per mm² when cell distribution is the question (standard IHC practice). Normalise cell based scores to the number of evaluable cells of that type, and density to measured viable tissue area (standard IHC practice). Use identical thresholds, retrieval and imaging settings across cases, and document excluded necrotic or damaged regions (page IHC retrieval rule; standard IHC practice).
Which findings support a true BCAS4 positive result rather than artefact?
A plausible result is reproducible cytoplasmic staining in intact cells, consistent with UniProt’s cytoplasmic assignment and HPA’s general cytoplasmic tissue profile (UniProt Q8TDM0 subcellular location; HPA tissue IHC profile). Inspect documented high staining cell populations, such as appendix glandular cells, but do not treat their HPA signal as definitive validation because HPA rates tissue IHC reliability Uncertain (HPA tissue IHC: High in appendix glandular cells; HPA tissue IHC reliability). Challenge staining confined to section edges, necrotic areas, unexpected nuclei or cell types outside the defined population using matched controls and morphology (UniProt Q8TDM0 subcellular location; standard IHC practice). Exclude endogenous peroxidase deposits with a no primary control and peroxidase blocking check before interpreting DAB as BCAS4 (standard IHC practice).
Boster reagents
Best BCAS4 / Breast carcinoma-amplified sequence 4 IHC Antibodies
A13963-1 has IHC and IF images from human breast carcinoma (catalog image captions). Human and mouse reactivity is listed, but the supplied images show human tissue only (catalog: reactivity; image captions).
Real IHC data
Anti-BCAS4 Antibody
Cat # A13963-1
A13963-1 will render with an IHC image of human breast carcinoma stained at 5 μg/mL (catalog IHC image caption). Its IF image shows human breast carcinoma tissue stained at 20 μg/mL (catalog IF image caption).
Which to pick: Choose A13963-1 for paraffin-section IHC (catalog: IHC-P application); its IHC caption documents human breast carcinoma, but does not report the fixative (catalog IHC image caption). For IF, A13963-1 has a human breast carcinoma tissue image; ICC is not separately listed (catalog: IF application; IF image caption). For mouse samples, A13963-1 lists mouse reactivity, although the supplied IHC and IF images show human tissue (catalog: reactivity; image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8TDM0 (BCAS4_HUMAN, Breast carcinoma-amplified sequence 4).
- Human Protein Atlas. BCAS4 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. BCAS4 subcellular location (ICC-IF): Localized to the cytosol and intermediate filaments..
- Human Protein Atlas. BCAS4 antibody validation summary (1 antibodies).
- RNF180 loss stabilizes BCAS4 to enhance PD-L1 expression and immune evasion in hepatocellular carcinoma. iScience 2026 — PMC13520631.
- PubMed PMID:12378525 — UniProt-cited evidence.
- PubMed PMID:11780052 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for BCAS4 (Breast carcinoma-amplified sequence 4). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A13963-1 at 5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (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 A13963-1); 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-BCAS4, 5 μg/mL (datasheet A13963-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Low agreement between antibody staining and RNA (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage is unspecified (UniProt); Regulation: Elevated in most breast cancer cell lines (UniProt). Evidence: UniProt Q8TDM0, HPA tissue IHC · Uncertain, HPA ICC-IF · cytosol and intermediate filaments., Datasheet A13963-1, PMC13520631. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/BCAS4/immunohistochemistry-ihc-guide.