CREB3L2 / Cyclic AMP-responsive element-binding protein 3-like protein 2 · IHC design guide

Design Immunohistochemistry for CREB3L2

Plan chromogenic IHC on paraffin sections with the catalog antibody at 2–5 μg/ml (datasheet A04591-1). Use strongly stained ciliary rootlets in bronchus or fallopian tube as reference tissue, and score cytoplasmic and membranous staining separately (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for CREB3L2 (IHC for CREB3L2): expected localisation Mainly cytoplasmic with some membranous staining (HPA tissue IHC), antibody A04591-1, validated IHC image, and IHC protocol steps — Printable **CREB3L2** IHC protocol sheet — expected localisation Mainly cytoplasmic with some membranous staining (HPA tissue IHC), antibody A04591-1, controls and protocol steps. Open the full CREB3L2 IHC guide →

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CREB3L2 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	Mainly cytoplasmic with some membranous staining (HPA tissue IHC)
	Staining pattern	Ciliary rootlets stain strongly in bronchus and fallopian tube (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A04591-1)
	Positive control ⓘ	Bronchus+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 paraffin sections. (standard IHC practice; not target-specific)
	Caveat	ER stress can shift detectable signal toward nuclei (UniProt)
	Regulation	ER stress may increase nuclear signal (UniProt)
	Isoform / epitope	3 isoforms; cleavage splits cytosolic/luminal epitopes (UniProt)

Section 1

Recommended CREB3L2 IHC & IF Protocols

The catalog antibody’s IHC-P protocol (datasheet: A04591-1) is accompanied by published CREB3L2 protocols for cartilage (PMC4952234) and breast cancer tissue (PMC11721608).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human thyroid cancer tissue; fixative not specified (datasheet A04591-1)
Fixation	Image fixative and duration unreported (datasheet A04591-1); 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 A04591-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A04591-1)
Primary antibody	Rabbit anti-CREB3L2, 2-5 μg/ml (datasheet A04591-1)
Primary incubation	Overnight at 4 °C (datasheet A04591-1)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A04591-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	CREB3L2-positive staining in ciliated cells (ciliary rootlets) of bronchus (HPA tissue IHC: High). HPA tissue profile: Mainly cytoplasmic expression with additional membranous expression. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA pH 8.0 for the catalog antibody (datasheet: A04591-1). Use the stated pretreatment when reproducing either published protocol (PMC4952234; PMC11721608).

Section 2

What Is the Expected CREB3L2 Staining Pattern?

CREB3L2 is an ER membrane protein with a cytosolic N-terminal region that can enter the nucleus after stress-induced cleavage (UniProt Q70SY1 topology and processing). In tissue IHC, expect mainly cytoplasmic staining with some membranous staining, especially strong signal at ciliary rootlets in bronchial and fallopian-tube ciliated cells (HPA: tissue IHC). HPA rates the tissue staining “Enhanced,” while reporting medium consistency with RNA data (HPA: tissue IHC reliability).

What am I looking at on my slide?
High staining at ciliary rootlets in bronchial or fallopian-tube ciliated cells, with mainly cytoplasmic and some membranous signal (HPA: tissue IHC).	This matches the clearest tissue pattern in the supplied IHC evidence (HPA: High in these ciliated cells). Score the named cell population and its subcellular pattern; whole-section intensity can obscure a localized rootlet signal (general IHC practice).
Some cells show nuclear signal alongside cytoplasmic signal (UniProt Q70SY1 processing; HPA: ICC-IF nucleoplasm).	Nuclear localization is biologically plausible because the cleaved N-terminal region enters the nucleus (UniProt Q70SY1 processing). Nuclear staining alone cannot establish ER stress or cleavage in a section; interpret it with the antibody’s epitope and appropriate controls (general IHC practice).
Strong staining appears chiefly in an unexpected compartment, such as extracellular material, rather than the cell-associated pattern (HPA: tissue IHC profile; UniProt Q70SY1 localization).	Treat this as a possible artefact until controls support specificity (general IHC practice). CREB3L2 has documented ER and nuclear localization, and the reported tissue IHC profile is mainly cytoplasmic with additional membranous expression (UniProt Q70SY1; HPA: tissue IHC).
Staining dominates a cell population reported as undetected, such as adipocytes, while expected positive cells stain weakly (HPA: Not detected in adipocytes; High in bronchial ciliated cells).	Consider cross-reactivity or endogenous detection activity, and inspect negative controls (general IHC practice). An HPA “Not detected” result describes that sampled cell population under its assay conditions; it does not prove universal absence (HPA: tissue IHC).
The chromogen is diffuse across cells and surrounding tissue, obscuring cell boundaries and ciliary rootlets (general IHC practice).	This limits compartment and cell-type scoring (general IHC practice). Review primary-antibody omission and detection controls for nonspecific background; the diffuse pattern does not match the localized ciliary-rootlet signal reported by HPA (HPA: tissue IHC).

💡Expected CREB3L2 appearanceCall a result positive when cell-associated, mainly cytoplasmic signal with possible membranous staining includes high ciliary-rootlet staining in bronchial or fallopian-tube ciliated cells; diffuse extracellular color or dominant staining in reported-negative cells warrants control review (HPA: tissue IHC; general IHC practice).

How each factor affects the staining
Membrane topology	CREB3L2 has one transmembrane segment at residues 380–400, with residues 1–379 cytoplasmic and 401–520 lumenal (UniProt Q70SY1 topology). Epitope position therefore matters when interpreting which protein forms an antibody could detect.
Stress-dependent processing	The ER-resident protein can move to the Golgi for cleavage; its released N-terminal region enters the nucleus (UniProt Q70SY1 processing). Nuclear signal is plausible, but staining by itself does not identify the cleaved form.
Tissue and cell selection	Use the reported high ciliary-rootlet signal as a reference pattern; kidney tubular cells and several glandular or neuronal populations are reported at medium intensity (HPA: tissue IHC). Compare named cells, not tissues as uniformly positive.
Antibody evidence	HPA015068 has Enhanced IHC evidence; HPA015534 has Supported ICC evidence, with no IHC status listed in this payload (HPA: antibodies). These statuses apply to their respective assays and do not establish identical staining across antibodies.
IF/ICC: where should signal appear?	HPA reports nucleoplasm as supported and ER as approved in ICC-IF images from BJ, Hep-G2 and U2OS cells (HPA: subcellular). Use that localization to interpret IF images; this IHC section supplies no IF protocol.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
No signal appears in bronchial ciliated cells expected to stain strongly (HPA: High in ciliary rootlets).	A failed detection step, unsuitable primary-antibody conditions or lost tissue antigen are possibilities (general IHC practice); absence alone does not distinguish them.	Check that the ciliated cells are present, examine a known-positive control processed with the run, and verify the antibody and detection steps against their supplied instructions (general IHC practice).
Nuclear staining is scored as automatically false (UniProt Q70SY1 processing; HPA: ICC-IF nucleoplasm).	That rule overlooks the documented nuclear destination of the cleaved N-terminal region (UniProt Q70SY1 processing).	Record nuclear and cytoplasmic compartments separately; use antibody epitope information and controls before assigning the nuclear signal to CREB3L2 processing (general IHC practice).
Broad, uniform color hides the expected ciliary-rootlet pattern (HPA: High in bronchial and fallopian-tube ciliary rootlets).	Excess nonspecific binding or detection background may obscure localized staining (general IHC practice).	Compare a primary-antibody omission control; review blocking, antibody concentration, washing and chromogen development using the chosen IHC workflow (general IHC practice).
Reported-negative cells show prominent color, including adipocytes or ovarian stroma cells (HPA: Not detected in these cells).	Cross-reactivity or endogenous detection activity is possible; HPA’s negative observations alone cannot identify the cause (HPA: tissue IHC; general IHC practice).	Check omission and detection controls, then compare cell-specific staining with a positive reference section before calling the unexpected signal specific (general IHC practice).
An apparently positive section shows only extracellular deposits or tissue edges (HPA: tissue IHC profile).	Localized deposition or edge-related background can mimic a positive chromogenic result (general IHC practice).	Inspect intact interior cells and controls; require a reproducible cell-associated cytoplasmic or membranous pattern before scoring the section positive (HPA: tissue IHC; general IHC practice).
Weak staining in a tissue is taken as proof that CREB3L2 is absent everywhere in it (HPA: low tissue specificity).	HPA reports levels by specific cell population, including low staining in hepatocytes and medium staining in kidney tubular cells (HPA: tissue IHC).	Score the relevant cell population and confirm assay performance with a positive reference section; report weak or undetected staining under the tested conditions (HPA: tissue IHC; general IHC practice).

Source: UniProt Q70SY1 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing CREB3L2 staining

IHC analysis of CREB3L2 using anti-CREB3L2 antibody (A04591-1). CREB3L2 was detected in a paraffin-embedded section of human thyroid cancer 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 2 μg/ml rabbit anti-CREB3L2 Antibody (A04591-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A04591-1 · IHCIHC analysis of CREB3L2 using anti-CREB3L2 antibody (A04591-1). CREB3L2 was detected in a paraffin-embedded section of human thyroid cancer

IF analysis of CREB3L2 using anti-CREB3L2 antibody (A04591-1). CREB3L2 was detected in an immunocytochemical section of U2OS cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-CREB3L2 Antibody (A04591-1) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A04591-1 · IFIF analysis of CREB3L2 using anti-CREB3L2 antibody (A04591-1). CREB3L2 was detected in an immunocytochemical section of U2OS cells. Enzyme a

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000182158-CREB3L2/tissue/cerebral+cortex#imid_3886896

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000182158-CREB3L2/tissue/colon#imid_3886830

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000182158-CREB3L2/tissue/liver#imid_3886839

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000182158-CREB3L2/tissue/kidney#imid_3886832

HPA ICC-IF BJ [Human fibroblast] — CREB3L2 (HPA015534)

HPA · ICC-IF BJ [Human fibroblast]Human Protein Atlas immunofluorescence, BJ [Human fibroblast], antibody HPA015534; green = CREB3L2, blue = nucleus, red = microtubules.

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

Sample controls for CREB3L2 IHC & IF

🧪Run bronchus first: ciliated cells must show ciliary-rootlet staining (HPA: High in bronchial ciliated cells). Use adipose tissue as the negative tissue: adipocytes should show no specific staining (HPA: Not detected in adipocytes); on the bronchus slide, adjacent nonciliated cells should lack the ciliary-rootlet pattern, though their overall CREB3L2 status is not established by these rows.

Positive control tissue: Bronchus (Ciliated cells (ciliary rootlets), HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CREB3L2 in BJ [Human fibroblast], Hep-G2, U2OS, with annotated localisation: Nucleoplasm (supported), Endoplasmic reticulum (approved) (HPA subcellular).

Technical controls: Include no-primary (secondary-only), concentration-matched rabbit IgG isotype, and a CREB3L2-knockout biological negative (selected-SKU IHC caption: rabbit primary antibody; standard IHC controls). For chromogenic bronchus sections, quench endogenous peroxidase and check that mucus or chromogen deposits do not mimic ciliary-rootlet staining (HPA: High in bronchial ciliated-cell ciliary rootlets; standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU IHC caption). The reported paraffin IHC used heat retrieval in EDTA at pH 8.0, but the supplied evidence does not establish whether retrieval is essential (selected-SKU IHC caption). No supplied evidence shows that frozen sections or IF are easier; bronchial ciliary-rootlet staining needs careful distinction from luminal deposits (HPA: High in bronchial ciliated-cell ciliary rootlets; standard IHC practice).

HPA tissue IHC evidence for CREB3L2

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.

Positive expression · recommended positive controls

Tissue	Cell type	Level	Evidence	Source
Bronchus	Ciliated cells (ciliary rootlets)	High	Protein (IHC)	HPA →
Fallopian tube	Ciliated cells (ciliary rootlets)	High	Protein (IHC)	HPA →
Adrenal gland	Glandular cells	Medium	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Medium	Protein (IHC)	HPA →
Cerebral cortex	Neuronal cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Appendix	Glandular cells	Not detected	Protein (IHC)	HPA →
Colon	Endothelial cells	Not detected	Protein (IHC)	HPA →
Duodenum	Glandular cells	Not detected	Protein (IHC)	HPA →
Endometrium	Cells in endometrial stroma	Not detected	Protein (IHC)	HPA →

Section 3

Advanced CREB3L2 IHC Tips

Troubleshoot CREB3L2 chromogenic IHC by checking retrieval, staining compartment, antibody epitope and cell type before interpreting signal intensity.

Which retrieval conditions should I start with for CREB3L2 in paraffin sections?

Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A04591-1). The catalog antibody's tissue image used this retrieval before incubation at 2 μg/ml overnight at 4°C (datasheet A04591-1). If staining is weak, compare retrieval duration on adjacent sections while holding antibody concentration and DAB development time constant (standard IHC practice). Check both cytoplasmic and membranous staining during that comparison, since those are the principal tissue patterns reported for CREB3L2 (HPA tissue IHC). The image caption identifies paraffin embedding but does not report a fixative, so it cannot establish retrieval performance across fixation conditions (datasheet A04591-1).

Could fixation explain weak or inconsistent CREB3L2 staining?

Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state its fixative or fixation duration (datasheet A04591-1). Record those variables for each specimen, then compare sections processed with matched retrieval, primary antibody incubation and DAB development (standard IHC practice). If signal varies between batches, include the same control section in each run and inspect morphology alongside staining before changing retrieval conditions (standard IHC practice). Do not infer a CREB3L2 fixation effect from its membrane topology, glycosylation sites or reported tissue staining; those observations provide no fixation comparison (UniProt Q70SY1; HPA tissue IHC).

Should CREB3L2 staining be cytoplasmic, membranous or nuclear?

CREB3L2 resides in the ER membrane and can yield a nuclear N-terminal fragment after stress-dependent cleavage (UniProt Q70SY1). Tissue IHC is reported as mainly cytoplasmic with additional membranous staining, while subcellular imaging reports nucleoplasm and ER localisation (HPA tissue IHC; HPA subcellular). Score cytoplasmic or membranous and nuclear DAB signals separately, recording the fraction of cells in each category rather than combining them into one intensity value (standard IHC practice). Interpret a shift toward nuclear staining alongside antibody epitope information and controls, because compartment alone does not establish that cleavage occurred (UniProt Q70SY1 processing; standard IHC practice).

How does epitope position affect interpretation of CREB3L2 staining?

CREB3L2 has 3 reported isoforms, a cytoplasmic region spanning residues 1–379, a transmembrane segment at 380–400 and a lumenal region at 401–520 (UniProt Q70SY1). Stress-dependent processing releases an N-terminal fragment that can enter the nucleus, so epitope position matters when interpreting nuclear DAB signal (UniProt Q70SY1 processing). Check the catalog antibody's stated immunogen or epitope before comparing nuclear and ER-associated staining; the supplied caption does not specify one (datasheet A04591-1). If its epitope remains unknown, report the observed compartments without assigning them to a particular isoform or processed fragment (standard IHC practice).

How can IF help check a puzzling CREB3L2 IHC pattern?

Use IF as a separate localisation check and multiplex CREB3L2 with a marker for the expected cell type, such as ciliated cells when examining bronchus or fallopian tube (HPA tissue IHC: High in ciliary rootlets). Choose a red or far-red fluorophore after inspecting an unstained section for tissue autofluorescence (standard IF practice). Plan permeabilisation around the antibody's epitope: residues 1–379 face the cytoplasm, whereas 401–520 are lumenal across the ER membrane (UniProt Q70SY1 topology). Because the catalog antibody's epitope is unspecified here, optimise access empirically and compare nuclear and ER-associated fluorescence with the chromogenic pattern, without treating IF as validation of the IHC result (datasheet A04591-1; standard IF practice).

How do I reduce diffuse DAB background without losing CREB3L2 signal?

Run a no-primary control to assess detection-system staining, and include an endogenous peroxidase block before HRP and DAB development (standard IHC practice). The catalog image used goat-serum blocking at 10%, primary antibody at 2 μg/ml overnight at 4°C, and a peroxidase-conjugated secondary for 30 minutes at 37°C (datasheet A04591-1). If background persists, compare primary concentration and wash conditions on adjacent sections while keeping DAB exposure matched (standard IHC practice). Judge any improvement against cell-associated cytoplasmic or membranous staining, the principal CREB3L2 tissue pattern, rather than against total section darkness (HPA tissue IHC).

What should I measure when comparing CREB3L2 IHC across samples? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring, then record the percentage of positive cells and an H-score for cytoplasmic or membranous staining (standard IHC practice; HPA tissue IHC profile). Score nuclear staining separately because CREB3L2 can produce a nuclear N-terminal fragment after ER-stress processing (UniProt Q70SY1 processing). For spatial comparisons, count positive cells per mm² of viable tissue or report positive cells as a percentage of the same annotated cell population (standard IHC practice). Normalise comparisons to the same tissue area or cell denominator, section-processing batch and DAB development conditions, and exclude necrotic or folded regions (standard IHC practice).

How can I distinguish convincing CREB3L2 staining from artefact?

Look for reproducible cell-associated cytoplasmic or membranous signal, the main reported tissue pattern, while assessing nuclear signal as a separate finding (HPA tissue IHC; UniProt Q70SY1). Strong staining at section edges, within necrosis or in a no-primary control calls for review of processing and detection background (standard IHC practice). Check cell identity: ciliated cells at bronchial or fallopian-tube rootlets are reported as high, whereas adipocytes are reported as not detected (HPA tissue IHC). A pattern confined to an unexpected compartment or cell population needs confirmation with controls and epitope information; endogenous peroxidase can also produce misleading DAB signal (standard IHC practice; UniProt Q70SY1 topology).

Boster reagents

Best CREB3L2 / Cyclic AMP-responsive element-binding protein 3-like protein 2 IHC Antibodies

The catalog antibody has IHC data from a human paraffin-embedded thyroid cancer section and IF/ICC data from U2OS cells (A04591-1 image captions).

Real IHC data

Anti-CREB3L2 Antibody ®

Cat # A04591-1

View product →

A04591-1 is the only listed SKU and is validated for human IHC on a paraffin-embedded thyroid cancer section (A04591-1 IHC caption; catalog applications and reactivity). Its IF/ICC image shows staining in U2OS cells (A04591-1 IF caption).

Which to pick: Choose A04591-1 for paraffin-section IHC: its image documents heat retrieval in EDTA at pH 8.0 and primary antibody at 2 μg/ml (A04591-1 IHC caption). The same SKU supports IF/ICC at 5 μg/ml in U2OS cells (A04591-1 IF caption); it is a rabbit antibody with no clone specified, and only human reactivity is listed, so there is no documented cross-species option (A04591-1 catalog). The IHC caption does not report the fixative (A04591-1 IHC caption).

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

References

UniProt Consortium. UniProt entry Q70SY1 (CR3L2_HUMAN, Cyclic AMP-responsive element-binding protein 3-like protein 2).
Human Protein Atlas. CREB3L2 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. CREB3L2 subcellular location (ICC-IF): Localized to the nucleoplasm and endoplasmic reticulum..
Human Protein Atlas. CREB3L2 antibody validation summary (2 antibodies).
The unfolded protein response genes in human osteoarthritic chondrocytes: PERK emerges as a potential therapeutic target. Arthritis research & therapy 2016 — PMC4952234.
Low-Grade Fibromyxoid Sarcoma and Related Subtypes: A Systematic Review and Pooled Analysis of 773 Cases. Cancers 2026 — PMC12896792.
Clinical and molecular characterization of primary sclerosing epithelioid fibrosarcoma of bone and review of the literature. Genes, chromosomes & cancer 2020 — PMC7082133.
Noncanonical UPR factor CREB3L2 drives immune evasion of triple-negative breast cancer through Hedgehog pathway modulation in T cells. Science advances 2025 — PMC11721608.
PubMed PMID:12915480 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.
PubMed PMID:12690205 — UniProt-cited evidence.