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

Design Immunohistochemistry for EBAG9

Plan EBAG9 staining in paraffin sections around the granular cytoplasmic pattern observed in tissue (HPA tissue IHC). The guide covers the catalog antibody’s IHC dilution of 0.5–1 μg/ml (datasheet PB9553) and how to assess staining against that pattern (HPA tissue IHC).

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 cytoplasm (HPA tissue IHC); Golgi membrane expected (UniProt), antibody PB9553, validated IHC image, and IHC protocol steps — Printable **EBAG9** IHC protocol sheet — expected localisation Granular cytoplasm (HPA tissue IHC); Golgi membrane expected (UniProt), 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 cytoplasm (HPA tissue IHC); Golgi membrane expected (UniProt)
	Staining pattern	Many cell types show 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 soluble form could alter staining, but its existence is uncertain (UniProt)
	Regulation	No staining regulator specified (UniProt)
	Isoform / epitope	2 isoforms; check whether the epitope lies outside the aa 7–27 anchor (UniProt)

Section 1

Recommended EBAG9 IHC & IF Protocols

The catalog antibody protocol uses EDTA retrieval (datasheet PB9553). Two published EBAG9 IHC protocols provide ovarian cancer and mouse bone examples (PMC2409481 methods; PMC10835455 methods).

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, pH 8.0 (datasheet PB9553); consider citrate, pH 6.0, if optimization is needed (PMC2409481 methods).

Section 2

What Is the Expected EBAG9 Staining Pattern?

EBAG9 should appear as granular cytoplasmic staining in many cell types, including glandular cells in appendix, cervix and colon (HPA tissue IHC: ubiquitous granular cytoplasmic expression; High in these cells). A Golgi-associated distribution is consistent with its membrane location and transmembrane topology (UniProt O00559: Golgi membrane; residues 7–27 transmembrane). Tissue IHC evidence is rated Enhanced for agreement with RNA expression (HPA tissue IHC: Enhanced).

What am I looking at on my slide?
Granular cytoplasmic signal in appendix or cervix glandular cells.	This matches the reported pattern and High staining in these cells (HPA tissue IHC: ubiquitous granular cytoplasmic expression; High in appendix and cervix glandular cells). Judge the cellular pattern alongside the counterstain; intensity alone cannot establish specificity (general IHC practice).
Predominantly nuclear or uniformly surface-rim staining, without cytoplasmic granules.	This conflicts with tissue IHC and Golgi localization (HPA tissue IHC: granular cytoplasm; HPA subcellular: Golgi apparatus). Review morphology and controls before scoring it as EBAG9. Its transmembrane segment alone does not establish a surface-rim IHC pattern (UniProt O00559: residues 7–27 transmembrane).
Strong staining mainly in cardiomyocytes, myocytes or adipocytes.	These cells have reported Low staining (HPA tissue IHC: cardiomyocytes, skeletal myocytes and adipocytes Low). Disproportionately strong signal raises concern for cross-reactivity or endogenous detection activity; compare a no-primary control and known-positive glandular cells (general IHC practice).
Diffuse color over cells and surrounding section, obscuring granules.	This is hard to reconcile with the reported granular cytoplasmic pattern (HPA tissue IHC). Background can arise from nonspecific binding or endogenous detection activity (general IHC practice). Assess the no-primary control and whether the signal follows tissue structures before assigning a cell-level score.
No signal in appendix glandular cells on an otherwise interpretable section.	Appendix glandular cells are reported High (HPA tissue IHC). An absent result warrants a technical check of retrieval, antibody dilution and detection (general IHC practice). One negative run does not establish that the sample lacks EBAG9.

💡Expected EBAG9 appearanceCall a positive result when glandular cells show distinct granular cytoplasmic staining, with High signal expected in appendix and cervix (HPA tissue IHC); dominant nuclear color or diffuse section-wide color is suspect against the reported pattern (HPA tissue IHC: granular cytoplasm; general IHC practice).

How each factor affects the staining
Compartment and topology (UniProt O00559; HPA subcellular)	EBAG9 is annotated at the Golgi membrane, with one transmembrane segment at 7–27 and a cytoplasmic region at 28–213 (UniProt O00559). A granular cytoplasmic IHC pattern fits the reported tissue observations (HPA tissue IHC); topology does not identify the antibody's epitope.
Tissue choice (HPA tissue IHC; UniProt O00559)	Appendix, cervix and colon glandular cells are reported High; cardiomyocytes and skeletal myocytes are Low (HPA tissue IHC). UniProt's tissue-specificity note lists colon among sites without expression, so treat colon as a source discrepancy rather than a definitive negative (UniProt O00559; HPA tissue IHC: colon glandular cells High).
Antibody evidence (HPA antibodies; HPA tissue IHC)	HPA021153 and HPA021154 have Enhanced IHC status, while CAB072810 has Supported IHC status (HPA antibodies). The tissue profile has Enhanced reliability for antibody staining and RNA agreement (HPA tissue IHC). These ratings support pattern interpretation; they do not validate an unspecified catalog antibody or its dilution.
IF/ICC Q&A: where should EBAG9 appear? (HPA subcellular)	In IF/ICC, a Golgi-associated signal is expected (HPA subcellular: Golgi apparatus, enhanced). Images are listed for HeLa, MCF-7 and U2OS (HPA subcellular). This is a localization cross-check, not an IHC-P staining protocol or evidence that every paraffin section will resolve the Golgi.
Isoforms and soluble-form claim (UniProt O00559)	UniProt lists 2 isoforms but supplies no isoform-specific staining pattern here (UniProt O00559). It also says a reported soluble form is uncertain (UniProt O00559). Do not assign diffuse extracellular color to shedding or infer which isoform an antibody detects without epitope evidence.
Antigen retrieval (general IHC practice)	Retrieval conditions can affect routine paraffin IHC performance (general IHC practice). No EBAG9-specific retrieval or fixation-sensitivity result is supplied, so select and assess conditions using the antibody's validated IHC-P instructions and tissue controls; do not infer an EBAG9-specific fixation effect.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Expected glandular-cell signal is absent (HPA tissue IHC: appendix glandular cells High).	Retrieval, dilution or detection may have failed (general IHC practice).	Run a known-positive section alongside the sample; check the antibody's IHC-P instructions, retrieval settings and detection reagents (general IHC practice). Interpret a sample negative only after the control stains as expected.
Brown color is widespread with little cellular detail.	Nonspecific binding or endogenous detection activity may obscure the granular pattern (general IHC practice; HPA tissue IHC: granular cytoplasm).	Compare a no-primary control, review blocking and washes, and confirm the detection system's endogenous-activity controls (general IHC practice). Score only cell-associated signal that remains distinguishable from background.
Signal is chiefly nuclear.	The compartment conflicts with cytoplasmic tissue staining and Golgi localization (HPA tissue IHC; HPA subcellular).	Check the counterstain and control sections, then reassess antibody specificity or detection background (general IHC practice). Do not count nuclear-only color as the expected EBAG9 pattern.
Muscle or adipose cells dominate the positive field.	These cells are reported Low; prominent staining may reflect cross-reactivity or background (HPA tissue IHC; general IHC practice).	Compare their signal with known-positive glandular cells and a no-primary control (HPA tissue IHC: appendix glandular cells High; general IHC practice). Reassess the result if the low-level cells remain strongest.
Colon stains strongly, but a reference note calls it absent.	The supplied sources disagree: colon glandular cells are High in HPA tissue IHC, while UniProt's tissue note lists colon as unexpressed (HPA tissue IHC; UniProt O00559).	Report the observed cell type and compartment with its source context. Use a second reported high-staining tissue, such as appendix, to check the run (HPA tissue IHC: appendix glandular cells High).
A diffuse extracellular deposit is interpreted as shed EBAG9.	UniProt describes the existence of a soluble form as uncertain (UniProt O00559).	Check whether the deposit persists in a no-primary control and whether cellular granules are present (general IHC practice; HPA tissue IHC: granular cytoplasm). Describe the deposit as observed; do not assign it to a soluble form from morphology alone.

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 and score its glandular cells for EBAG9 staining (HPA: High in appendix glandular cells). HPA detects EBAG9 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and assess nonglandular cells on the same slide for background without assuming they are EBAG9-negative (HPA: no negative tissue rows).

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 control and a concentration-matched rabbit immunoglobulin isotype control for the rabbit primary; a validated EBAG9 knockout specimen or specific immunizing-peptide block can provide a biological specificity control (caption: rabbit anti-EBAG9 PB9553; standard IHC practice). For appendix sections, check endogenous peroxidase and biotin background when using the caption’s streptavidin–biotin/DAB detection system (caption: SABC with DAB; standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected PB9553 paraffin-section caption does not state the fixative (caption: fixative unreported). The reported mouse-testis IHC used heat retrieval in EDTA, pH 8.0; test whether that condition works for appendix rather than treating retrieval dependence as established (caption: EDTA retrieval; HPA: High in appendix glandular cells). For IF/ICC, Golgi localization and images in HeLa, MCF-7 and U2OS support a localization check, but the supplied evidence does not establish whether IF or frozen sections are easier than paraffin IHC; assess tissue autofluorescence if using IF (HPA: Golgi apparatus, ICC-IF images; standard IF 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, tissue preservation, cellular pattern and controls before comparing scores.

What should I change when EBAG9 staining is weak after retrieval?

Start with heat-mediated retrieval in EDTA, pH 8.0, for paraffin sections (PB9553 tissue-IHC caption). The documented mouse testis workflow then used 1 μg/ml primary antibody overnight at 4°C, so check those conditions before changing retrieval (PB9553 tissue-IHC caption). If staining remains weak, compare a small retrieval-time series while keeping section thickness, antibody concentration and detection constant (standard IHC practice). Include a section processed with the documented conditions in each run to reveal run-to-run variation (standard IHC practice). Inspect tissue morphology and background alongside signal, because excessive heating can damage sections and make an apparent gain difficult to interpret (standard IHC practice).

How can I assess whether fixation is reducing EBAG9 signal?

EBAG9-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (PB9553 tissue-IHC caption). Record fixative, fixation duration, tissue thickness and processing history for each specimen, then compare matched sections under identical retrieval and detection conditions (standard IHC practice). Use the documented EDTA, pH 8.0 retrieval and 1 μg/ml primary concentration as a starting point for that comparison (PB9553 tissue-IHC caption). Check whether reduced staining tracks a processing batch rather than a tissue region, and inspect morphology before changing retrieval intensity (standard IHC practice). Do not assign any observed fixation effect specifically to EBAG9 without a controlled comparison (standard IHC practice).

Is diffuse or membrane-like staining plausible for EBAG9?

Expect a granular cytoplasmic pattern in tissue sections (HPA tissue IHC profile), consistent with Golgi localisation (HPA subcellular; UniProt O00559). Score that pattern separately from diffuse cytoplasmic, surface or nuclear colour, and compare each pattern with a no-primary control (standard IHC practice). EBAG9 has a transmembrane segment at residues 7–27 and a cytoplasmic region at 28–213 (UniProt O00559 topology). That topology alone does not establish that a particular stain at the cell surface is specific in paraffin sections (UniProt O00559 topology; standard IHC interpretation). If the expected granular pattern disappears while diffuse colour increases, review retrieval, antibody concentration and detection background together (standard IHC practice).

Could isoforms or epitope accessibility explain discordant staining?

EBAG9 has two annotated isoforms, but the supplied evidence does not map the catalog antibody’s epitope to either one (UniProt O00559; PB9553 tissue-IHC caption). Check the antibody’s epitope documentation before interpreting a negative section as absence of both isoforms (standard IHC interpretation). Residues 36, 41 and 94 are annotated phosphorylation sites in the cytoplasmic region (UniProt O00559 modified residues and topology). Their presence does not establish that phosphorylation changes this antibody’s staining, because epitope and modification sensitivity are unspecified (UniProt O00559; PB9553 tissue-IHC caption). Compare matched sections with fixed retrieval and detection settings, and report epitope uncertainty when staining patterns disagree (standard IHC practice).

How should I investigate a discordant EBAG9 immunofluorescence result?

Treat IF as a separate assay: the selected antibody evidence is a paraffin-section chromogenic workflow, with no fixative stated in its caption (PB9553 tissue-IHC caption). For multiplex IF, pair EBAG9 with a marker identifying the expected cell population, such as glandular cells where that population is being assessed (HPA tissue IHC profile; standard IF practice). Choose fluorophores after examining unstained tissue autofluorescence, and include single-label controls to assess channel spillover (standard IF practice). If the antibody recognises an epitope in the cytoplasmic 28–213 region, optimise permeabilisation to expose it; the supplied evidence does not locate this antibody’s epitope (UniProt O00559 topology; PB9553 tissue-IHC caption). Compare any IF pattern with Golgi localisation, without assuming that IF conditions transfer to IHC (HPA subcellular; standard assay interpretation).

How do I distinguish EBAG9 staining from chromogenic background?

The documented workflow blocked with 10% goat serum, used biotinylated goat anti-rabbit secondary for 30 minutes at 37°C, and developed with a streptavidin–biotin complex and DAB (PB9553 tissue-IHC caption). Include no-primary and secondary-only controls to assess nonspecific detection, and check for endogenous biotin with a suitable control when using this detection system (standard IHC practice). Apply a peroxidase block before DAB detection and keep development time consistent across comparison sections (standard chromogenic IHC practice). Assess background in unstained areas and across cell types before increasing primary dilution or shortening incubation (standard IHC practice). A widespread brown haze without granular cytoplasmic enrichment is less convincing than the reported tissue pattern (HPA tissue IHC profile; standard IHC interpretation).

What scoring method best captures EBAG9 staining differences? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring: the reported tissue pattern is granular cytoplasmic (HPA tissue IHC profile). For each region, record the percentage of positive target cells and staining intensity, then calculate an H-score if intensity categories are reproducible (standard IHC scoring practice). Normalise positive-cell counts to the number of evaluable target cells, or stained area to evaluable tissue area in mm², rather than to the whole image (standard IHC quantification practice). Keep retrieval, DAB development, imaging and thresholds constant across groups (standard IHC practice). Report cell type and region with each score because glandular cells and several other populations show differing reported staining levels (HPA tissue IHC profile).

What makes an EBAG9-positive section convincing?

A convincing result shows granular cytoplasmic staining in intact cells, matching the reported tissue pattern and Golgi localisation (HPA tissue IHC profile; HPA subcellular). Judge cell identity explicitly: glandular cells in appendix, cervix, colon, duodenum and epididymis have reported high staining (HPA tissue IHC profile). Nuclear-only colour or staining confined to tissue edges or necrotic regions needs control-based review before it is scored as EBAG9 (HPA subcellular; standard IHC interpretation). Compare no-primary and detection controls to identify endogenous enzyme activity or reagent background in the DAB workflow (standard chromogenic IHC practice). Interpret a negative result against specimen quality and assay controls, particularly because the selected caption does not report fixation (PB9553 tissue-IHC caption; standard IHC interpretation).

Boster reagents

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

PB9553 has real IHC images of paraffin sections from human prostatic cancer and mouse and rat testis (PB9553 IHC image captions).

Real IHC data

Anti-EBAG9 Antibody ®

Cat # PB9553

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

Which to pick: Choose PB9553 for tissue IHC in paraffin sections; its listed IHC dilution is 0.5–1 μg/ml, and the fixative is unreported (catalog: PB9553 IHC dilution; PB9553 IHC image captions). For work across human, mouse and rat samples, PB9553 has listed reactivity and IHC images for all three species (catalog: PB9553 reactivity; PB9553 IHC image captions). No IF/ICC antibody can be recommended from this catalog entry: PB9553 lists IHC and WB, has no IF image or dilution, and reports no clone (catalog: PB9553 applications, IF images, IF dilution and clone).

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.