EBAG9 / Receptor-binding cancer antigen expressed on SiSo cells · IHC design guide

Design Immunohistochemistry for EBAG9

Plan EBAG9 paraffin-section IHC using its granular cytoplasmic tissue pattern (HPA tissue IHC). This guide covers fixation consistency and interpretation, including epitope assessment for 2 isoforms (UniProt).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for EBAG9 (IHC for EBAG9): expected localisation Granular cytoplasmic staining (HPA tissue IHC), antibody PB9553, validated IHC image, and IHC protocol steps — Printable **EBAG9** IHC protocol sheet — expected localisation Granular cytoplasmic staining (HPA tissue IHC), antibody PB9553, controls and protocol steps. Open the full EBAG9 IHC guide →

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EBAG9 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	Granular cytoplasmic staining (HPA tissue IHC)
	Staining pattern	Widespread granular cytoplasmic staining (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet PB9553)
	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	A reported soluble form remains uncertain (UniProt)
	Regulation	Staining intensity regulators are not specified (UniProt; HPA tissue IHC)
	Isoform / epitope	2 isoforms; check epitope coverage and topology (UniProt)

Section 1

Recommended EBAG9 IHC & IF Protocols

The catalog antibody protocol uses EDTA retrieval at pH 8.0 (datasheet). The published IHC protocols below provide conditions reported for EBAG9 staining (PMC2409481; PMC10835455; PMC10278705).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded mouse testis tissue; fixative not specified (datasheet PB9553)
Fixation	Image fixative and duration unreported (datasheet PB9553); 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 PB9553); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet PB9553)
Primary antibody	Rabbit anti-EBAG9, 0.5-1μg/ml (datasheet PB9553)
Primary incubation	Overnight at 4 °C (datasheet PB9553)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9553)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	EBAG9-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression with a granular pattern. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet). The ovarian cancer study used citrate at pH 6.0 with a different antibody (PMC2409481).

Section 2

What Is the Expected EBAG9 Staining Pattern?

EBAG9 should appear as granular cytoplasmic staining in many cell types, with a Golgi-associated distribution (HPA tissue IHC: ubiquitous granular cytoplasmic expression; HPA ICC-IF: Golgi apparatus). HPA rates its tissue pattern Enhanced, reflecting high consistency with RNA expression (HPA tissue IHC: Enhanced). A Golgi membrane location is consistent with its single transmembrane segment at residues 7–27 (UniProt O00559 topology).

What am I looking at on my slide?
Granular cytoplasmic signal in appendix or colon glandular cells.	This matches reported High staining in those cells (HPA tissue IHC: appendix and colon glandular cells, High). Assess granularity and cell identity alongside intensity; the tissue label alone does not establish specificity (standard IHC practice).
Predominantly nuclear staining, or a crisp cell-surface rim without granular cytoplasm.	These patterns diverge from the reported granular cytoplasm and Golgi location (HPA tissue IHC; HPA ICC-IF). Consider nonspecific detection or a scoring error, and review controls before calling EBAG9 positive (standard IHC practice).
Strong staining in an unexpected cell population, with expected cells unstained.	Interpret the result against cell-level reference patterns; HPA reports High signal in cerebellar Purkinje cells and Low signal in heart cardiomyocytes (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible; investigate with appropriate controls (standard IHC practice).
Uniform haze across cells, stroma, and blank areas.	A widespread haze obscures the reported granular cytoplasmic pattern (HPA tissue IHC). Background can arise from excess antibody, incomplete blocking, or detection chemistry (standard IHC practice); it cannot be scored as cell-specific EBAG9 staining.
No signal in a known-positive tissue section.	Absence of signal in a reported High population, such as appendix glandular cells, calls assay performance into question (HPA tissue IHC: appendix glandular cells, High). Check section quality, controls, retrieval, and detection before interpreting an experimental sample as negative (standard IHC practice).

💡Expected EBAG9 appearanceCall a section positive when identifiable glandular or neuronal cells show granular cytoplasmic staining, potentially concentrated near the Golgi; High staining is reported in appendix glands and cerebellar Purkinje cells (HPA tissue IHC; HPA ICC-IF). Diffuse haze or isolated nuclear staining is suspect (standard IHC practice).

How each factor affects the staining
Compartment and topology	EBAG9 is annotated at the Golgi membrane, with one transmembrane segment at 7–27 and a cytoplasmic region at 28–213 (UniProt O00559 topology). This supports checking cytoplasmic distribution; topology alone does not identify the antibody epitope or predict retrieval needs.
Choice of reference tissue	HPA reports High glandular staining in colon and duodenum (HPA tissue IHC), although UniProt's tissue-specificity note says EBAG9 was not detected in colon or small intestine (UniProt O00559 tissue specificity). Treat the difference as a source discrepancy; use the observed HPA IHC pattern as the slide reference.
Antibody validation	The HPA tissue profile is Enhanced, and HPA021153 and HPA021154 have Enhanced IHC status; CAB072810 has Supported IHC status (HPA tissue IHC; HPA antibodies). These statuses support interpretation of reported patterns but do not validate an unlisted antibody or establish a working dilution.
Isoforms and possible soluble protein	UniProt lists 2 isoforms and notes a proposed soluble form whose existence is uncertain (UniProt O00559 isoforms; subcellular location). Neither annotation establishes which form an IHC antibody detects, so do not attribute diffuse or extracellular staining to shedding without further evidence.
What should IF/ICC show?	Golgi localization is reported as enhanced, with images listed for HeLa, MCF-7, and U2OS (HPA ICC-IF). Use that localization to compare compartments; IF/ICC has its own guide and no IF protocol is specified here.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Reported positive glands show no chromogenic signal.	The assay may have failed; HPA reports High staining in appendix glandular cells (HPA tissue IHC). The record does not establish an EBAG9-specific fixation effect.	Confirm positive-control staining and reagent function; check the IHC-validated antibody's documented retrieval and detection conditions (standard IHC practice).
Signal is mostly nuclear.	Nuclear dominance conflicts with granular cytoplasmic tissue staining and Golgi localization (HPA tissue IHC; HPA ICC-IF).	Compare a positive-control section and inspect background controls; score only cell-associated staining consistent with the reference pattern (standard IHC practice).
A cell-surface rim dominates the section.	A Golgi membrane protein need not yield a surface-rim IHC pattern (UniProt O00559 subcellular location; HPA ICC-IF: Golgi).	Recheck compartment assignment at higher magnification and compare with granular cytoplasmic staining in a reference positive cell population (HPA tissue IHC; standard IHC practice).
Strong signal appears across stroma and expected low-staining cells.	Nonspecific or endogenous detection activity may be contributing (standard IHC practice); HPA reports Low staining in soft-tissue fibroblasts and adipocytes (HPA tissue IHC).	Run appropriate negative and detection controls, then reassess blocking and detection chemistry (standard IHC practice).
The whole section has diffuse brown haze.	Excess reagent or incomplete background control can obscure cell-level granularity (standard IHC practice; HPA tissue IHC: granular cytoplasmic pattern).	Inspect a no-primary control, adjust blocking and washing, and reassess antibody concentration using documented IHC-P conditions (standard IHC practice).
Colon glands stain despite a UniProt tissue note suggesting absence.	The sources differ: HPA reports High colon glandular staining, while UniProt reports no detection in colon (HPA tissue IHC; UniProt O00559 tissue specificity).	Record the source discrepancy and judge the section using its cell-level pattern and controls; avoid treating either source alone as proof of assay failure (standard IHC practice).

Source: UniProt O00559 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing EBAG9 staining

IHC analysis of EBAG9 using anti-EBAG9 antibody (PB9553). EBAG9 was detected in a paraffin-embedded section of mouse testis tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 μg/ml rabbit anti-EBAG9 Antibody (PB9553) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

PB9553 · IHCIHC analysis of EBAG9 using anti-EBAG9 antibody (PB9553). EBAG9 was detected in a paraffin-embedded section of mouse testis tissue. Heat med

IHC analysis of EBAG9 using anti-EBAG9 antibody (PB9553). EBAG9 was detected in a paraffin-embedded section of rat testis tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 μg/ml rabbit anti-EBAG9 Antibody (PB9553) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

PB9553 · IHCIHC analysis of EBAG9 using anti-EBAG9 antibody (PB9553). EBAG9 was detected in a paraffin-embedded section of rat testis tissue. Heat media

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000147654-EBAG9/tissue/cerebral+cortex#imid_5577268

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000147654-EBAG9/tissue/colon#imid_5564666

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000147654-EBAG9/tissue/liver#imid_5563435

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000147654-EBAG9/tissue/kidney#imid_5563436

HPA · ICC-IF HeLaHuman Protein Atlas immunofluorescence, HeLa, antibody CAB072810; green = EBAG9, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for EBAG9 IHC & IF

🧪Run appendix first; its glandular cells should stain (HPA: High in appendix glandular cells). HPA detects EBAG9 in all 45 scored tissues (HPA: no negative tissue listed), so use no-primary and isotype controls for the negative comparison; non-glandular cells on the positive slide should be assessed for background rather than assumed to be EBAG9-negative.

Positive control tissue: Appendix (Glandular cells, HPA High)

Negative control tissue: None in HPA: EBAG9 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 EBAG9 in HeLa, MCF-7, U2OS, with annotated localisation: Golgi apparatus (enhanced) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only section, a matched rabbit IgG isotype control (PB9553 caption: rabbit primary), and EBAG9-knockout tissue if available as a biological negative. Quench endogenous peroxidase and check for endogenous biotin background in the appendix when using biotin-based DAB detection (PB9553 caption: biotinylated secondary, SABC and DAB).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected PB9553 paraffin-section caption does not state a fixative (PB9553 caption). That caption documents heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required or that frozen sections are easier (PB9553 caption). ICC-IF images show enhanced Golgi localization in HeLa, MCF-7 and U2OS cells (HPA: subcellular), but provide no basis to call IF easier; in appendix sections, inflammatory cells or blood can contribute endogenous peroxidase background during DAB detection (standard IHC practice).

HPA tissue IHC evidence for EBAG9

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 →
Cerebellum	Purkinje cells	High	Protein (IHC)	HPA →
Cerebral cortex	Neuronal cells	High	Protein (IHC)	HPA →
Cervix	Glandular cells	High	Protein (IHC)	HPA →
Colon	Glandular cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
None in HPA: EBAG9 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced EBAG9 IHC Tips

Troubleshoot EBAG9 staining in paraffin sections by checking retrieval, compartment-specific signal, controls, and how staining is scored (datasheet PB9553; HPA tissue IHC).

What should I change if EBAG9 staining is weak after antigen retrieval?

Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet PB9553). The documented mouse testis workflow then used 10% goat serum and 1 μg/ml primary antibody overnight at 4°C (datasheet PB9553). If signal remains weak, compare retrieval heating and cooling consistency across sections before changing antibody concentration (standard IHC practice). Use a section from the documented tissue as a run control, and compare granular cytoplasmic staining rather than total brown area (datasheet PB9553; HPA tissue IHC). If retrieval damages morphology, shorten the heating step while keeping EDTA at pH 8.0 as the reference condition (standard IHC practice; datasheet PB9553).

Could fixation explain weak or uneven EBAG9 staining?

EBAG9-specific sensitivity to fixation is unknown because the selected paraffin-section caption does not state a fixative (datasheet PB9553). Record the specimen’s fixative, fixation duration, and processing history before comparing staining across cases (standard IHC practice). Keep section thickness and the documented EDTA pH 8.0 retrieval condition consistent when assessing whether weak staining tracks with processing (standard IHC practice; datasheet PB9553). Examine intact tissue and section edges separately, since uneven preservation can complicate interpretation of a granular cytoplasmic pattern (standard IHC practice; HPA tissue IHC). Test any proposed fixation change on matched material before treating a signal difference as altered EBAG9 expression (standard IHC practice).

Is diffuse cytoplasmic or surface staining plausible for EBAG9?

Expect predominantly granular cytoplasmic staining in tissue sections, consistent with the reported Golgi localisation (HPA tissue IHC; HPA subcellular; UniProt O00559 localisation). EBAG9 has a transmembrane segment at residues 7–27 and a cytoplasmic region at 28–213 (UniProt O00559 topology). Compare suspicious diffuse or rim staining with the granular pattern in a control section before assigning it to EBAG9 (HPA tissue IHC; standard IHC practice). A proposed soluble form is uncertain, so diffuse extracellular DAB alone is insufficient evidence for secreted EBAG9 (UniProt O00559 localisation). Check whether the suspected signal persists in the no-primary control and follows tissue structures rather than folds or edges (standard IHC practice).

How could isoforms or epitope placement affect EBAG9 IHC?

EBAG9 has 2 annotated isoforms, but the supplied evidence does not map the catalog antibody’s epitope to either one (UniProt O00559 isoforms; datasheet PB9553). Its transmembrane segment spans residues 7–27, while residues 28–213 are assigned to the cytoplasmic side (UniProt O00559 topology). Consequently, do not infer isoform coverage or epitope accessibility from a positive chromogenic section alone (standard IHC interpretation; UniProt O00559 isoforms). Phosphorylation is annotated at residues 36, 41, and 94, but its effect on this antibody’s staining is unreported (UniProt O00559 modified residues; datasheet PB9553). If staining differs across specimens, first compare retrieval and controls, then seek an epitope mapping result before attributing the difference to an isoform or modification (standard IHC practice).

How can IF help assess an ambiguous EBAG9 IHC pattern?

Use IF as a companion check of compartment and cell identity when chromogenic staining is ambiguous (standard IF practice; HPA subcellular). Multiplex EBAG9 with a validated glandular cell marker where glandular cells are being assessed, since several glandular populations show high tissue staining (HPA tissue IHC; standard IF practice). Choose a fluorophore away from the specimen’s strongest autofluorescence and inspect a no-primary channel control before calling puncta positive (standard IF practice). For an antibody recognising the cytoplasmic region at residues 28–213, include permeabilisation; the catalog antibody’s epitope side is not supplied (UniProt O00559 topology; standard IF practice; datasheet PB9553). Compare any IF puncta with Golgi localisation, while retaining the documented EDTA pH 8.0 condition as the IHC reference (HPA subcellular; datasheet PB9553).

What causes widespread brown staining in EBAG9 sections?

First compare the stained slide with a no-primary control to identify signal from detection reagents or endogenous tissue activity (standard IHC practice). The documented workflow uses a biotinylated secondary, streptavidin–biotin complex, and DAB, so assess endogenous biotin and peroxidase background when brown signal is widespread (datasheet PB9553; standard IHC practice). A peroxidase block belongs to the general chromogenic workflow; it is not evidence that EBAG9 requires a special block (standard IHC practice). Also check whether the 10% goat serum block and 1 μg/ml primary condition were followed before increasing primary concentration (datasheet PB9553). Treat uniform staining of debris, folds, or tissue edges as suspect unless it resolves into the reported granular cytoplasmic pattern (standard IHC practice; HPA tissue IHC).

How should EBAG9 staining be scored across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring; EBAG9 tissue staining is reported as granular cytoplasmic (HPA tissue IHC). For a cell based comparison, record the percentage of positive cells and a prespecified intensity grade, then calculate an H-score if graded intensity is reproducible (standard IHC practice). For spatial comparisons, report positive cells per mm² of evaluable tissue and exclude folds, necrosis, and poorly preserved areas (standard IHC practice). Normalise counts to the relevant cell population or evaluable tissue area, and keep retrieval and DAB development consistent across slides (standard IHC practice; datasheet PB9553). Report control performance and the scoring threshold alongside results, because threshold changes can alter the apparent positive fraction (standard IHC practice).

How do I distinguish true EBAG9 staining from artefact?

Give most weight to staining in intact cells that forms a granular cytoplasmic pattern compatible with Golgi localisation (HPA tissue IHC; HPA subcellular). Assess the expected cell population explicitly; high staining is reported in appendix and colon glandular cells, whereas several muscle cell populations show low staining (HPA tissue IHC). Signal confined to section edges, necrotic areas, or folds should be checked against nearby preserved tissue and the no-primary control (standard IHC practice). Brown deposits that persist without primary antibody can reflect endogenous peroxidase or detection-system background in a DAB workflow (standard IHC practice; datasheet PB9553). Because the proposed soluble form is uncertain, do not interpret extracellular deposits alone as proof of EBAG9 secretion (UniProt O00559 localisation).

Boster reagents

Best EBAG9 / Receptor-binding cancer antigen expressed on SiSo cells IHC Antibodies

The IHC-validated anti-EBAG9 antibody PB9553 has images from paraffin sections of mouse and rat testis and human prostatic cancer (catalog IHC captions); no IF images are supplied (catalog).

Real IHC data

Anti-EBAG9 Antibody ®

Cat # PB9553

View product →

PB9553 is listed for IHC in human, mouse and rat (catalog applications and reactivity). Its IHC captions show paraffin sections of mouse and rat testis and human prostatic cancer (PB9553 IHC captions).

Which to pick: For tissue IHC, choose PB9553: its paraffin-section images use 1 μg/ml antibody with EDTA retrieval at pH 8.0 (PB9553 IHC captions). No SKU here lists IF/ICC or supplies IF images, so an IF/ICC choice is unsupported (catalog applications and IF image alts). For cross-species IHC, PB9553 lists human, mouse and rat reactivity and has an image for each species; its host is rabbit, while clonality and tissue fixative are unreported (catalog reactivity and host; PB9553 IHC captions).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry O00559 (RCAS1_HUMAN, Receptor-binding cancer antigen expressed on SiSo cells).
Human Protein Atlas. EBAG9 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. EBAG9 subcellular location (ICC-IF): Localized to the Golgi apparatus..
Human Protein Atlas. EBAG9 antibody validation summary (5 antibodies).
Expression of EBAG9/RCAS1 is associated with advanced disease in human epithelial ovarian cancer. British journal of cancer 2004 — PMC2409481.
EBAG9-deficient mice display decreased bone mineral density with suppressed autophagy. iScience 2024 — PMC10835455.
Reevaluation of the 22-1-1 antibody and its putative antigen, EBAG9/RCAS1, as a tumor marker. BMC cancer 2005 — PMC1164403.
Single-cell RNA sequencing reveals the transcriptomic landscape of kidneys in patients with ischemic acute kidney injury. Chinese medical journal 2023 — PMC10278705.
PubMed PMID:10426319 — UniProt-cited evidence.
PubMed PMID:9418891 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.